Postintegration HIV-1 infection of cervical epithelial cells mediates contact-dependent productive infection of T cells.

The female genital epithelium plays a protective role against invading pathogens; however, sexual transmission of human immunodeficiency virus type 1 (HIV-1) still occurs in healthy women. To model virus-cell interactions in this barrier during sexual transmission, we studied the uptake and infection of ectocervical and endocervical cell lines with cell-free fluorescent protein-expressing recombinant HIV-1 carrying primary transmitted/founder envelope genes. We observed that a subset of both the ectocervical and endocervical epithelial cells become productively infected with cell-free HIV-1 in a CD4-independent manner. In addition, the ability of the semen-derived enhancer of virus infection (SEVI) to enhance virus-epithelial cell interactions was studied. This infection is increased approximately 2-5 fold when inoculation occurs in the presence of SEVI fibrils. Once infected, the epithelial cells are capable of transmitting the virus to target CD4 T cells in coculture in a contact-dependent manner that uses conventional CD4- and coreceptor-dependent entry. The infection of target CD4 T cells only occurs when de novo HIV-1 is produced within the epithelial cells. These findings suggest that a subset of cervical epithelial cells may be actively involved in establishing a systemic HIV infection and should be a target when designing prevention strategies to protect against HIV-1 sexual transmission.

Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission.

Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.

Modulation of osteoblast gap junction connectivity by serum, TNFalpha, and TRAIL.

We studied the effects of serum growth factors and of TNF family proteins on osteoblast gap junction connectivity. Serum starvation of human MG63 osteosarcoma cells or nontransformed osteoblasts decreased connexin43 protein. TNFalpha or TRAIL reduced connexin43 further. Serum starvation redistributed gap junctions but did not reduce intercellular diffusion. In contrast, TNFalpha or TRAIL reduced gap junctions on cell processes and decreased intercellular diffusion. Effects of TNFs on connexin43 were mediated by lysosomal proteolysis. Activating analogs of cAMP increased connexin43 protein, but did not block effects of serum starvation, TNFalpha, or TRAIL on connexin43 protein. Connexin43 and connectivity recovered overnight if stimuli were withdrawn. Surprisingly, connexin43 mRNA increased in serum starvation and with TNFalpha or TRAIL. Since beta-catenin is a binding partner of connexin43, when connexin43 is degraded, beta-catenin activation may contribute to a reflexive increase in connexin43 transcription. We conclude that osteoblast connectivity is regulated by a multifactorial system that maintains intercellular connections. Serum starvation, TNFalpha and TRAIL augmented connexin43 degradation and connexin43 transcription. Cell-cell communication was maintained in serum starvation, which may model response to acute injury, but was sensitive to TNFs. These inflammatory agents mediated selective, reversible removal of connexin43 from cell processes.

Platelet-derived growth factor receptor-alpha: a novel therapeutic target in human hepatocellular cancer.

Hepatocellular cancer (HCC) is a disease of poor prognosis. Identifying novel molecular aberrations might present opportunities to identify new therapeutic targets. Due to the similarities between the processes of development and cancer, we used early developing livers to identify genes that might play a primary role in HCC. Platelet-derived growth factor receptor-alpha (PDGFRalpha) was identified from microarray using early developing mouse livers. Expression of PDGFRalpha and its upstream effectors, PDGF-AA and PDGF-CC, were examined in HCC tissues (n = 43) by Western blot, real-time PCR, and immunohistochemistry. Finally, effect of anti-PDGFRalpha antibody (mAb 3G3, ImClone Systems, Inc.) was examined on human hepatoma cells. A high expression of PDGFRalpha was observed during early liver development. HCCs (17 of 21) revealed cytoplasmic PDGFRalpha and activated PDGFRalpha (phospho-Tyr(754)) by immunohistochemistry. Additional HCCs (14 of 22) showed elevated PDGFRalpha levels when compared with the adjacent normal livers by Western blots. Of these 14 patients, 3 showed increased PDGFRalpha gene expression, 3 showed elevated PDGF-AA, and 4 had higher PDGF-CC levels in the tumors compared with adjacent livers. Multiple hepatoma cell lines, when treated with mAb 3G3, showed significant decreases in cell proliferation and survival (P < 0.05). In conclusion, approximately 70% of HCC tissues had elevated PDGFRalpha levels due to diverse mechanisms. PDGFRalpha inhibition in hepatoma cells led to diminution of tumor cell survival and proliferation and thus might be of therapeutic significance.

beta-Catenin is critical for early postnatal liver growth.

The Wnt/beta-catenin pathway plays an important role in embryonic liver development, morphogenesis, and organogenesis. Here, we report on the activation of beta-catenin during early postnatal liver growth. Modulation of beta-catenin expression was studied in CD-1 mice livers over a time course of 0 to 30 postnatal days (PD) and 3 mo. Increases in total and active beta-catenin were observed in developing livers from PD 5 to 20. A concomitant increase in the beta-catenin-transcription factor (TCF) complex along with nuclear and cytoplasmic beta-catenin was also evident, which coincided with ongoing hepatocyte proliferation by PCNA immunohistochemistry. This activation of beta-catenin was multifactorial, including cyclical inhibition of glycogen synthase kinase-3beta, suppression of casein kinase-IIalpha, and a transient increase in beta-catenin gene expression. Coprecipitation experiments revealed the formation of the beta-catenin-cadherin complex at PD 5, whereas adequate beta-catenin-c-Met complex at the hepatocyte membrane did not form until PD 20, which might be contributing to the free beta-catenin pool during early postnatal growth. Furthermore, beta-catenin liver-specific knockout mice exhibited smaller livers at PD 30, secondary to diminished hepatocyte proliferation. These data indicate that the activation of beta-catenin is critical for early postnatal liver growth and development.

R-Etodolac decreases beta-catenin levels along with survival and proliferation of hepatoma cells.

BACKGROUND/AIMS: Inhibition of hepatoma cells by cyclooxygenase (COX)-2-dependent and -independent mechanisms has been shown previously. Here, we examine the effect of Celecoxib, a COX-2-inhibitor and R-Etodolac, an enantiomer of the nonsteroidal anti-inflammatory drug Etodolac, which lacks COX-inhibitory activity, on the Wnt/beta-catenin pathway and human hepatoma cells. METHODS: Hep3B and HepG2 cell lines were treated with Celecoxib or R-Etodolac, and examined for viability, DNA synthesis, Wnt/beta-catenin pathway components, and downstream target gene expression. RESULTS: Celecoxib at high doses affected beta-catenin protein by inducing its degradation via GSK3beta and APC along with diminished tumor cell proliferation and survival. R-Etodolac at physiological doses caused decrease in total and activated beta-catenin protein secondary to decrease in its gene expression and post-translationally through GSK3beta activation. In addition, increased beta-catenin-E-cadherin was also observed at the membrane. An associated inhibition of beta-catenin-dependent Tcf reporter activity, decreased levels of downstream target gene products glutamine synthetase and cyclin-D1, and decreased proliferation and survival of hepatoma cells was evident. CONCLUSIONS: The antitumor effects of Celecoxib (at high concentrations) and R-Etodolac (at physiological doses) on HCC cells were accompanied by the down-regulation of beta-catenin demonstrating a useful therapeutic strategy in hepatocellular cancer.

Activation of Wnt/beta-catenin pathway during hepatocyte growth factor-induced hepatomegaly in mice.

Hepatocyte growth factor (HGF) and beta-catenin both play a crucial role in stimulating hepatocyte proliferation, but whether these 2 pathways cooperate in inducing hepatocyte proliferation is unclear. We have previously reported that beta-catenin forms a complex with c-Met (HGF receptor) that undergoes dissociation because of beta-catenin tyrosine phosphorylation on stimulation by HGF. It is also known that delivery of the human HGF gene cloned in a plasmid under a CMV promoter results in hepatomegaly in mice. In addition, recently characterized beta-catenin transgenic mice also showed hepatomegaly. The present study was based on the hypothesis that HGF-induced hepatomegaly is mediated, at least in part, by activation of the Wnt/beta-catenin pathway. Here we report that delivery of the human HGF gene delivery in mice led to hepatomegaly via beta-catenin activation in the liver in 1- and 4-week studies. The mechanisms of beta-catenin activation in the 1-week study included loss of c-Met-beta-catenin association as well as canonical beta-catenin activation, leading to its nuclear translocation. In the 4-week study, beta-catenin activation was observed via canonical mechanisms, whereas the c-Met-beta-catenin complex remained unchanged. In both studies there was an associated increase in the E-cadherin-beta-catenin association at the membrane. In addition, we generated liver-specific beta-catenin knockout mice, which demonstrated significantly smaller livers. HGF gene delivery failed to induce hepatomegaly in these beta-catenin conditionally null mice. In conclusion, beta-catenin- and HGF-mediated signaling pathways cooperate in hepatocyte proliferation, which may be crucial in liver development, regeneration following partial hepatectomy, and pathogenesis of hepatocellular carcinoma.

Tyrosine residues 654 and 670 in beta-catenin are crucial in regulation of Met-beta-catenin interactions.

beta-Catenin, a key component of the canonical Wnt pathway, is also regulated by tyrosine phosphorylation that regulates its association to E-cadherin. Previously, we reported its association with the hepatocyte growth factor (HGF) receptor Met at the membrane. HGF induced Met-beta-catenin dissociation and nuclear translocation of beta-catenin, which was tyrosine-phosphorylation-dependent. Here, we further investigate the Met-beta-catenin interaction by selectively mutating several tyrosine residues, alone or in combination, in beta-catenin. The mutants were subcloned into FLAG-CMV vector and stably transfected into rat hepatoma cells, which were treated with HGF. All single or double-mutant-transfected cells continued to show HGF-induced nuclear translocation of FLAG-beta-catenin except the mutations affecting 654 and 670 simultaneously (Y654/670F), which coincided with the lack of formation of beta-catenin-TCF complex and DNA synthesis, in response to the HGF treatment. In addition, the Y654/670F-transfected cells also showed no phosphorylation of beta-catenin or dissociation from Met in response to HGF. Thus, intact 654 and 670 tyrosine residues in beta-catenin are crucial in HGF-mediated beta-catenin translocation, activation and mitogenesis.

Aberrant Wnt/beta-catenin signaling in pancreatic adenocarcinoma.

Wnt/beta-catenin signaling plays an important role in normal development. However, its aberrant activation is associated with several cancers. The aim of this study is to examine the Wnt/beta-catenin pathway in patients with advanced pancreatic adenocarcinoma (n = 31). Paraffin sections from tumors (n = 16) and normal pancreata (n = 3) were used to determine the localization of beta-catenin. An additional 15 frozen tumors, adjacent normal pancreata (n = 5), or normal pancreata (n = 4) were utilized for protein isolation. Tumors were also examined for mutations in exon 3 of the CTNNB1 gene. More than 65% of the tumors showed an increase in total beta-catenin, consistent with its enhanced membranous, cytoplasmic, and nuclear localization, but only two showed mutations in CTNNB1. The majority of the remaining tumors demonstrated concurrent increases in Wnt-1 and frizzled-2 (positive regulators) and a decrease in Ser45/Thr41-phospho-beta-catenin. Electrophoretic mobility shift assay demonstrated beta-catenin-T-cell factor binding in tumors only. Adenomatous polyposis coli and axin, which are both negative regulators, remained unchanged. Unexpectedly, total glycogen synthase kinase-3beta protein was elevated in these tumors. Elevated levels of E-cadherin were also observed, although E-cadherin-beta-catenin association in tumors remained unaffected. Thus, Wnt/beta-catenin activation was observed in 65% of pancreatic adenocarcinomas, independently of beta-catenin gene mutations in most tumors.

beta-Catenin regulation during matrigel-induced rat hepatocyte differentiation.

Hepatocytes in primary cultures de-differentiate and re-differentiate following addition of Engelbreth-Holm-Swarm mouse sarcoma (matrigel) to the cultures. The Wnt/beta-catenin pathway has been shown to be important in liver growth and development. Here, we investigate changes in beta-catenin and its mechanism, during matrigel-induced hepatocyte differentiation. Primary rat hepatocytes were cultured for 8 days, and matrigel was added to half of the cultures. Total and nuclear protein and total RNA were extracted at different days of culture and examined for beta-catenin and other Wnt pathway components. A significant increase in total beta-catenin protein was observed upon matrigel addition, during hepatocyte differentiation, despite a decrease in beta-catenin and frizzled-1 (Wnt receptor) expression. A concurrent decrease in the glycogen synthase kinase-3beta (GSK3beta), axin, and ser45/thr41-phosphorylated beta-catenin proteins was observed in matrigel-treated cultures, implying decreased degradation of beta-catenin. Interestingly, a decrease in nuclear beta-catenin and total active beta-catenin was observed in the presence of matrigel. Matrigel also induced an increased association of beta-catenin with Met (hepatocyte growth factor receptor), whereas association with E-cadherin remained unchanged. This coexisted with decreased beta-catenin tyrosine phosphorylation. Thus, beta-catenin undergoes multifactorial regulation during matrigel-induced hepatocyte differentiation and maturation; this induces its stabilization and membrane translocation, possibly contributing to hepatocyte differentiation.

Mouse fetal liver cells in artificial capillary beds in three-dimensional four-compartment bioreactors.

Bioreactors containing porcine or adult human hepatocytes have been used to sustain acute liver failure patients until liver transplantation. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and viability of adult cells in vitro. We investigated the use of fetal hepatocytes as an alternative cell source in bioreactors. Mouse fetal liver cells from gestational day 17 possessed intermediate differentiation and function based on their molecular profile. When cultured in a three-dimensional four-compartment hollow fiber-based bioreactor for 3 to 5 weeks these cells formed neo-tissues that were characterized comprehensively. Albumin liberation, testosterone metabolism, and P450 induction were demonstrated. Histology showed predominant ribbon-like three-dimensional structures composed of hepatocytes between hollow fibers. High positivity for proliferating cell nuclear antigen and Ki-67 and low positivity for terminal dUTP nick-end labeling indicated robust cell proliferation and survival. Most cells within these ribbon arrangements were albumin-positive. In addition, cells in peripheral zones were simultaneously positive for alpha-fetoprotein, cytokeratin-19, and c-kit, indicating their progenitor phenotype. Mesenchymal components including endothelial, stellate, and smooth muscle cells were also observed. Thus, fetal liver cells can survive, proliferate, differentiate, and function in a three-dimensional perfusion culture system while maintaining a progenitor pool, reflecting an important advance in hepatic tissue engineering.

Epidermal growth factor receptor: a novel target of the Wnt/beta-catenin pathway in liver.

BACKGROUND & AIMS: Wnt/beta-catenin activation is observed in normal liver development, regeneration, and liver cancer. Our aim was to elucidate the regulation and mechanism of this pathway in liver. METHODS: We report the generation and characterization of liver-specific nonmutated beta-catenin-overexpressing transgenic mice. Transgenic livers were examined for their morphology and phenotype by histology, proliferation, apoptosis, and microarray analysis. RESULTS: Transgenic livers displayed a significant increase in cytoplasmic, membranous, and nuclear beta-catenin in hepatocytes as compared with their wild-type littermates, which display a predominant membranous localization only. A 15%-20% increase in the liver weight-body weight ratio was evident in transgenic mice secondary to increased hepatocyte proliferation. Microarray analysis showed differential expression of approximately 400 genes in the transgenic livers. Epidermal growth factor receptor RNA and protein and increased levels of activated epidermal growth factor receptor and Stat3 were observed in the transgenic livers. Epidermal growth factor receptor promoter analysis showed a T-cell factor-binding site, and subsequent reporter assay confirmed epidermal growth factor receptor activation in response to Wnt-3A treatment that was abrogated by frizzled related protein 1, a known Wnt antagonist. Epidermal growth factor receptor inhibition successfully decreased liver size in transgenic mice. Next, 7 of 10 hepatoblastomas displayed simultaneous beta-catenin and epidermal growth factor receptor up-regulation, thus suggesting a strong relationship between these 2 proteins in tumors. CONCLUSIONS: beta-Catenin transgenic mice show an in vivo hepatotrophic effect secondary to increased basal hepatocyte proliferation. Epidermal growth factor receptor seems to be a direct target of the pathway, and epidermal growth factor receptor activation might contribute toward some mitogenic effects of increased beta-catenin in liver: epidermal growth factor receptor inhibition might be useful in such states.

Morpholino oligonucleotide-triggered beta-catenin knockdown compromises normal liver regeneration.

BACKGROUND/AIMS: Wnt/beta-catenin activation is seen during early liver regeneration (LR) observed as stabilization and translocation to the nucleus followed by an overall decrease. However, beta-catenin continues to be in hepatocyte nucleus and membrane, secondary to its increased gene expression at 6-72 h. METHODS: In the present study, we examined the effect of ablating beta-catenin transcription on LR. Twelve male fisher rats were subjected to two-third partial hepatectomy followed by administration of beta-catenin antisense phospho-morpholino oligonucleotide (AS) in six or mismatch control (CON) injection in the remaining 6 via superior mesenteric vein. Three animals from each group were sacrificed at 24 h and 7 days for liver assessment. RESULTS: AS group exhibited a significant decrease in total beta-catenin at 24 h. A significant decrease in liver/body weight ratio was also observed in the AS group at 24 h and 7 days that was due to decreased proliferation. Among the targets of this pathway c-myc and uPAR levels showed significant decrease while cyclin-D1 remained unaffected. CONCLUSIONS: We demonstrate the importance of beta-catenin in early liver regeneration especially in hepatocyte proliferation. Also, c-myc and uPAR might be crucial downstream effectors of beta-catenin during liver regeneration.

Fibroblast growth factor enriches the embryonic liver cultures for hepatic progenitors.

Fibroblast growth factors (FGFs) play an important role in hepatic induction during development. The aim of our study was to investigate the effect of exogenous FGFs on ex vivo liver development. We begin our analysis by examining FGF signaling during early mouse liver development. Phospho-FGF receptor (Tyr653/654) was detected in embryonic day 10 (E10) to E12 livers only. Next, E10 livers were cultured in the presence of FGF1, FGF4, or FGF8 for 72 hours and examined for histology, proliferation, apoptosis, and differentiation. FGFs especially FGF8 promoted sheet-like architecture, cell proliferation, and survival as compared to the control. All FGFs induced a striking increase in the number of c-kit and alpha-fetoprotein-positive progenitors, without altering albumin staining. However these progenitors were CK-19-positive (biliary and bipotential progenitor marker) only in the presence of FGF1 or FGF4 and not FGF8. FGFs also induced beta-catenin, a stem cell renewal factor in these cultures. In conclusion, the presence of activated FGFR indicates a physiological role of FGF during early liver development. FGF1 and FGF4 enrich the embryonic liver cultures for bipotential hepatic progenitors. FGF8 promotes such enrichment and induces a one-step differentiation toward a unipotential hepatocyte progenitor. Thus, FGFs might be useful for enrichment and propagation of developmental hepatic progenitors.

Beta-catenin is temporally regulated during normal liver development.

BACKGROUND & AIMS: beta-Catenin, a key component of the Wnt pathway, plays an important role in unregulated liver growth in liver tumors, in regulated growth during liver regeneration, and in ex vivo embryonic liver cultures. METHODS: We used developing livers from several stages of gestational development to examine beta-catenin expression, protein-protein interactions, localization, and regulation in prenatal and postnatal livers. RESULTS: Microarray, Northern, and protein analyses showed peak expression of beta-catenin during early liver development at Embryonic day 10 (E10)-E12, followed by a decrease and a complete loss of normal beta-catenin (97-kilodalton species) after E16 through the remaining prenatal period. At the early stages, beta-catenin localized to the cytoplasm and nuclei of resident cells in addition to its normal membranous localization, which was seen at all later stages and in adult liver. Decreases in beta-catenin levels at E14 onward coincided with its decreased gene expression and increased degradation, as seen by an increase in serine 45/threonine 41-phosphorylated beta-catenin and its other negative regulators, such as axin, adenomatous polyposis coli gene product (APC), and glycogen synthase kinase-3 beta. Finally, we showed an intact association of E-cadherin and beta-catenin despite the loss of beta-catenin at E16-E18, owing to the presence of membrane-associated smaller-molecular-weight beta-catenin species. CONCLUSIONS: We also identified a stage-specific expression and regulation of beta-catenin during liver development that might be crucial for physiological liver development. Nuclear and cytoplasmic beta-catenin corresponded to cell proliferation in liver development. Finally, a smaller-molecular-weight species of beta-catenin might be maintaining normal interactions at the membrane.

Wnt impacts growth and differentiation in ex vivo liver development.

The Wnt-beta-catenin pathway plays a role in liver growth and development. Here, we investigate the direct effect of Wnt-3A on ex vivo liver development. Livers from mouse embryos at day 10 were cultured in serum-free Wnt-3A-conditioned media alone or with HGF and insulin for 72 h and analyzed for histology, proliferation, apoptosis and lineage. Control cultures grown in serum-free conditions or Wnt-3A and sFRP-1 combination display loss of architecture and proliferation and increased apoptosis. In the presence of Wnt-3A, embryonic liver cultures show CK-19-positive cells (biliary phenotype) displaying proliferation, minimal apoptosis and duct-like histological arrangement. HGF and Wnt combination exhibited normal histology as seen in the presence of 10% serum displaying stem cells, hepatocytes and primitive bile ducts. HGF, insulin and Wnt combination provided no additional benefits rather had an overall deleterious effect. Thus, Wnt supports biliary differentiation by enhancing stem cell specification, hepatocyte trans-differentiation and promoting biliary survival. HGF and Wnt combination supports stem cells, hepatocytes and bile ducts. The addition of insulin to the combination of HGF and Wnt provided no growth or differentiation advantage. Our results indicate usefulness of Wnt and HGF in hepatocyte cultures and suggest their balance during normal liver development.