Biomedical subjects

Jean S Campbell

Publications and source records attributed to Jean S Campbell.

14 recordsLinked to original sources

PubMed2007

Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation.

UNLABELLED: Oval cells are hepatocytic precursors that proliferate in late-stage cirrhosis and that give rise to a subset of human hepatocellular carcinomas. Although liver regeneration typically occurs through replication of existing hepatocytes, oval cells proliferate only when hepatocyte proliferation is inhibited. Transforming growth factor-beta (TGF-beta) is a key inhibitory cytokine for hepatocytes, both in vitro and in vivo. Because TGF-beta levels are elevated in chronic liver injury when oval cells arise, we hypothesized that oval cells may be less responsive to the growth inhibitory effects of this cytokine. To examine TGF-beta signaling in vivo in oval cells, we analyzed livers of rats fed a choline-deficient, ethionine-supplemented (CDE) diet for phospho-Smad2. Phospho-Smad2 was detected in more than 80% of hepatocytes, but staining was substantially reduced in oval cells. Ki67 staining, in contrast, was significantly more common in oval cells than hepatocytes. To understand the inverse relationship between TGF-beta signaling and proliferation in oval cells and hepatocytes, we examined TGF-beta signaling in vitro. TGF-beta caused marked growth inhibition in primary hepatocytes and the AML12 hepatocyte cell line. Two oval cell lines, LE/2 and LE/6, were less responsive. The greater sensitivity of the hepatocytes to TGF-beta-induced growth inhibition may result from the absence of Smad6 in these cells. CONCLUSION: Our results indicate that oval cells, both in vivo and in vitro, are less sensitive to TGF-beta-induced growth inhibition than hepatocytes. These findings further suggest an underlying mechanism for the proliferation of oval cells in an environment inhibitory to hepatocytic proliferation.

Animals↗

PubMed2006

Sorting nexin 1 down-regulation promotes colon tumorigenesis.

PURPOSE: Colon cancer is one of the most common human malignancies, yet studies have only begun to identify the multiple mechanisms that underlie the development of this tumor. In this study, we have identified a novel mechanism, dysregulation of endocytic sorting, which promotes colon cancer development. EXPERIMENTAL DESIGN: Immunohistochemical and microarray analyses were done on human colon cancer tissue specimens to determine the levels of one endocytic protein, sorting nexin 1 (SNX1). SW480 cells, a human colon cancer cell line that retains a relatively high level of SNX1 expression, were used to assess the effects of down-regulating this protein by small hairpin RNA. Activation of signal transduction cascades was evaluated in these cells using Western blotting, and multiple functional assays were done. RESULTS: We determined by immunohistochemistry that the level of SNX1 was significantly down-regulated in 75% of human colon cancers. In corroborative studies using microarray analysis, SNX1 message was significantly decreased (log(2) ratio less than -1) for 8 of 19 colon carcinomas. Cell lines with reduced SNX1 levels showed increased proliferation, decreased apoptosis, and decreased susceptibility to anoikis. They also showed increased activation of epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 in response to epidermal growth factor. This increased activation was abolished by inhibition of endocytosis. CONCLUSIONS: These data suggest that loss of SNX1 may play a significant role in the development and aggressiveness of human colon cancer, at least partially through the mechanism of increased signaling from endosomes. Further, these findings suggest that dysregulation of endocytic proteins may represent a new paradigm in the process of carcinogenesis.

Apoptosis↗

PubMed2006

Reciprocal and coordinate regulation of serum amyloid A versus apolipoprotein A-I and paraoxonase-1 by inflammation in murine hepatocytes.

OBJECTIVE: During inflammation, the serum amyloid A (SAA) content of HDL increases, whereas apolipoprotein A-I (apoA-I) and paraoxonase-1 (PON-1) decrease. It remains unclear whether SAA physically displaces apoA-I or if these changes derive from coordinated but inverse transcriptional regulation of the HDL apolipoprotein genes. Because cytokines stimulate the hepatic expression of inflammatory markers, we investigated their role in regulating SAA, apoA-I, and PON-1 expression. METHODS AND RESULTS: A cytokine mixture (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, and IL-6) simultaneously induced SAA and repressed apoA-I and PON-1 expression levels. These effects were partially inhibited in cells pretreated with either nuclear factor kappaB (NF-kappaB) inhibitors (pyrrolidine dithiocarbamate, SN50, and overexpression of super-repressor inhibitor kappaB) or after exposure to the peroxisome proliferator-activated receptor-alpha (PPARalpha) ligands (WY-14643 and fenofibrate). Consistent with these findings, the basal level of SAA was increased, whereas apoA-I and PON-1 decreased in primary hepatocytes from PPARalpha-deficient mice as compared with wild-type mice. Moreover, neither WY-14643 nor fenofibrate had any effect on SAA, apoA-I, or PON-1 expression in the absence of PPARalpha. CONCLUSIONS: These results suggest that cytokines increase the expression of SAA through NF-kappaB transactivation, while simultaneously decreasing the expression of apoA-I and PON-1 by inhibiting PPARalpha activation. Inflammation may convert HDL de novo into a more proatherogenic form by coordinate but inverse transcriptional regulation in the liver, rather than by physical displacement of apoA-I by SAA.

Animals↗

PubMed2006

Proinflammatory cytokine production in liver regeneration is Myd88-dependent, but independent of Cd14, Tlr2, and Tlr4.

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Liver regeneration.

During liver regeneration after partial hepatectomy, normally quiescent hepatocytes undergo one or two rounds of replication to restore the liver mass by a process of compensatory hyperplasia. A large number of genes are involved in liver regeneration, but the essential circuitry required for the process may be categorized into three networks: cytokine, growth factor and metabolic. There is much redundancy within each network, and intricate interactions exist between them. Thus, loss of function from a single gene rarely leads to complete blockage of liver regeneration. The innate immune system plays an important role in the initiation of liver regeneration after partial hepatectomy, and new cytokines and receptors that participate in initiation mechanisms have been identified. Hepatocytes primed by these agents readily respond to growth factors and enter the cell cycle. Presumably, the increased metabolic demands placed on hepatocytes of the regenerating liver are linked to the machinery needed for hepatocyte replication, and may function as a sensor that calibrates the regenerative response according to body demands. In contrast to the regenerative process after partial hepatectomy, which is driven by the replication of existing hepatocytes, liver repopulation after acute liver failure depends on the differentiation of progenitor cells. Such cells are also present in chronic liver diseases, but their contribution to the production of hepatocytes in those conditions is unknown. Most of the new knowledge about the molecular and cellular mechanisms of liver regeneration is both conceptually important and directly relevant to clinical problems.

Animals↗

PubMed2005

Platelet-derived growth factor C induces liver fibrosis, steatosis, and hepatocellular carcinoma.

Members of the platelet-derived growth factor (PDGF) ligand family are known to play important roles in wound healing and fibrotic disease. We show that both transient and stable expression of PDGF-C results in the development of liver fibrosis consisting of the deposition of collagen in a pericellular and perivenular pattern that resembles human alcoholic and nonalcoholic fatty liver disease. Fibrosis in PDGF-C transgenic mice, as demonstrated by staining and hydroxyproline content, is preceded by activation and proliferation of hepatic stellate cells, as shown by collagen, alpha-smooth muscle actin and glial fibrillary acidic protein staining and between 8 and 12 months of age is followed by the development of liver adenomas and hepatocellular carcinomas. The hepatic expression of a number of known profibrotic genes, including type beta1 TGF, PDGF receptors alpha and beta, and tissue inhibitors of matrix metalloproteinases-1 and -2, increased by 4 weeks of age. Increased PDGF receptor alpha and beta protein levels were associated with activation of extracellular regulated kinase-1 and -2 and protein kinase B. At 9 months of age, PDGF-C transgenic mice had enlarged livers associated with increased fibrosis, steatosis, cell dysplasia, and hepatocellular carcinomas. These studies indicate that hepatic expression of PDGF-C induces a number of profibrotic pathways, suggesting that this growth factor may act as an initiator of fibrosis. Moreover, PDGF-C transgenic mice represent a unique model for the study of hepatic fibrosis progressing to tumorigenesis.

Animals↗

PubMed2005

Differential regulation of rodent hepatocyte and oval cell proliferation by interferon gamma.

Hepatocytes and intrahepatic progenitor cells (oval cells) have similar responses to most growth factors but rarely proliferate together. Oval cells constitute a reserve compartment that is activated when hepatocyte proliferation is inhibited. Interferon gamma (IFN-gamma) increases in liver injury that involves oval cell responses, but it is not upregulated during liver regeneration after partial hepatectomy. Based on these observations, we used well-characterized lines of hepatocytes (AML-12 cells) and oval cells (LE-6 cells) to investigate the potential mechanisms that regulate differential growth responses in hepatocytes and oval cells. We show that IFN-gamma blocks hepatocyte proliferation in vivo, and that in combination with either tumor necrosis factor (TNF) or lipopolysaccharide (LPS), it causes cell cycle arrest in hepatocytes but stimulates oval cell proliferation in cultured cells. The hepatocyte cell cycle arrest is reversible, is p53-independent, and is not associated with apoptosis. Treatment of AML-12 hepatocytes with IFN-gamma/LPS or IFN-gamma/TNF, but not with individual cytokines, induced NO synthase and generated NO, while similarly treated oval cells produced little if any NO. Generation of NO by an NO donor reproduced the inhibitory effect of the cytokine combinations on AML-12 cell replication, while NO inhibitors abolish the replication deficiency. In conclusion, we propose that IFN-gamma, in conjunction with TNF or LPS, can both inhibit hepatocyte proliferation through the generation of NO and stimulate oval cell replication. The response of hepatocytes and oval cells to cytokine combinations may contribute to the differential proliferation of these cells in hepatic growth processes.

Animals↗

PubMed2005

Inhibition of RIP2/RIck/CARDIAK activity by pyridinyl imidazole inhibitors of p38 MAPK.

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase (MAPK) have been used extensively in vitro and in vivo to investigate the role of p38 in physiological processes. As with other pharmacological inhibitors, non-specific targets of the p38 inhibitors have been reported. We have found that the protein kinase receptor interacting protein-2 (RIP2) is another target for the family of p38 inhibitors. The autophosphorylation of RIP2 was inhibited in vitro by the p38 inhibitors SB220025, SB203580 and PD169316 at concentrations comparable to those used to inhibit p38. We also identified two new in vitro substrates for RIP2, myelin basic protein and histone H3 with apparent Km values of 2.1 microM and 0.65 microM, respectively. The ability of RIP2 to phosphorylate these two substrates was sensitive to the p38 inhibitors as well. As was shown for p38alpha, a conserved threonine in the kinase domain of RIP2 is required for sensitivity to the inhibitors, indicating that the mechanism of inhibition of RIP2 is similar to that of p38. These results demonstrate that the pyridinyl imidazole inhibitors block RIP2 as well as p38 kinase activity.

Cell Line↗

PubMed2004

Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration.

The mechanisms that regulate the transition between the initial priming phase and DNA replication in liver regeneration are poorly understood. To study this transition, we compared events occurring after standard two-thirds partial hepatectomy, which elicits full regeneration, with response to a reduced hepatectomy, one-third partial hepatectomy (1/3PH), which leads to little DNA replication. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between the two procedures, cell cycle progression was significantly blunted in 1/3PH mice. Among the main defects observed in 1/3PH mice were an almost complete deficiency in retinoblastoma phosphorylation and the lack of increase in kinase activity associated with cyclin E. We report that, in two-thirds partial hepatectomy mice, the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) preceded the start of DNA replication and was not detectable in 1/3PH animals. Injection of HB-EGF into 1/3PH mice resulted in a >15-fold increase in DNA replication. Moreover, we show that hepatocyte DNA replication was delayed in HB-EGF knock-out mice. In summary, we show that HB-EGF is a key factor for hepatocyte progression through G(1)/S transition during liver regeneration.

Animals↗

PubMed2004

Epidermal growth factor receptor transactivation mediates tumor necrosis factor-induced hepatocyte replication.

Tumor necrosis factor (TNF) has multiple biological effects such as participating in inflammation, apoptosis, and cell proliferation, but the mechanisms of its effects on epithelial cell proliferation have not been examined in detail. At the early stages of liver regeneration, TNF functions as a priming agent for hepatocyte replication and increases the sensitivity of hepatocytes to growth factors such as transforming growth factor alpha (TGFalpha); however, the mechanisms by which TNF interacts with growth factors and enhances hepatocyte replication are not known. Using the AML-12 hepatocyte cell line, we show that TNF stimulates proliferation of these cells through transactivation of the epidermal growth factor receptor (EGFR). The transactivation mechanism involves the release of TGFalpha into the medium through activation of the metalloproteinase TNFalpha-converting enzyme (also known as ADAM 17). Binding of the ligand to EGFR initiates a mitogenic cascade through extracellular signal-regulated kinases 1 and 2 and the partial involvement of protein kinase B. TNF-induced release of TGFalpha and activation of EGFR signaling were inhibited by TNFalpha protease inhibitor-1, an agent that interferes with TNFalpha-converting enzyme activity. We suggest that TNF-induced transactivation of EGFR may provide an early signal for the entry of hepatocytes into the cell cycle and may integrate proliferative and survival pathways at the start of liver regeneration.

ADAM Proteins↗

PubMed2003

The role of hepatocytes and oval cells in liver regeneration and repopulation.

The liver has the unique capacity to regulate its growth and mass. In rodents and humans, it grows rapidly after resection of more than 50% of its mass. This growth process, as well as that following acute chemical injury is known as liver regeneration, although growth takes place by compensatory hyperplasia rather than true regeneration. In addition to hepatocytes and non-parenchymal cells, the liver contains intra-hepatic "stem" cells which can generate a transit compartment of precursors named oval cells. Liver regeneration after partial hepatectomy does not involve intra or extra-hepatic (hemopoietic) stem cells but depends on the proliferation of hepatocytes. Transplantation and repopulation experiments have demonstrated that hepatocytes, which are highly differentiated and long-lived cells, have a remarkable capacity for multiple rounds of replication. In this article, we review some aspects of the regulation of hepatocyte proliferation as well as the interrelationships between hepatocytes and oval cells in different liver growth processes. We conclude that in the liver, normally quiescent differentiated cells replicate rapidly after tissue resection, while intra-hepatic precursor cells (oval cells) proliferate and generate lineage only in situations in which hepatocyte proliferation is blocked or delayed. Although bone marrow stem cells can generate oval cells and hepatocytes, transdifferentiation is very rare and inefficient.

Animals↗

PubMed2003

Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes.

Cultured human hepatocytes have broad research and clinical applications; however, the difficulties in culturing rodent and human hepatocytes are well known. These problems include the rapid loss of the hepatocytic phenotype in primary culture and the limited replicating capacity of the cultured cells. We describe the establishment of serum-free primary cultures of human fetal hepatocytes (HFHs) that retain hepatocytic morphology and gene expression patterns for several months and maintain sufficient proliferative activity to permit subculturing for at least 2 passages. Initially, HFH cultures contained 2 main cell types that morphologically resembled large and small hepatocytes. The fetal hepatocytes expressed alpha-fetoprotein (AFP), cytokeratin (CK) 19, albumin, and other hepatic proteins. Treatment of the cultures with oncostatin M (OSM) increased cell size and enhanced cell differentiation and formation of bile canaliculi, probably through an effect on hepatocyte nuclear factor (HNF) 4alpha. Approximately 1 month after plating, multiple clusters of very small cells became apparent in the cultures. These cells had very few organelles and are referred to as blast-like cells. Flow cytometric analysis of these cells showed that they express oval cell/stem cell markers such as CD90 (Thy-1), CD34, and OV-6 but do not stain with antibodies to beta(2)-microglobulin. HFH cultures maintained for 9 to 12 months produced grossly visible organoids containing ductular structures that stained for CK18, CK19, and AFP. In conclusion, HFH cultures, which might contain a population of hepatic stem cells, constitute an excellent tool for a variety of studies with human hepatocytes, including the mechanisms of viral infection.

Biomarkers↗

PubMed2002

Cell culture model for acetaminophen-induced hepatocyte death in vivo.

Overdose of the popular, and relatively safe, analgesic acetaminophen (N-acetyl-p-aminophenol, APAP, paracetamol) can produce a fatal centrilobular liver injury. APAP-induced cell death was investigated in a differentiated, transforming growth factor alpha (TGFalpha)-overexpressing, hepatocyte cell line and found to occur at concentrations, and over time frames, relevant to clinical overdose situations. Coordinated multiorganellar collapse was evident during APAP-induced cytotoxicity with widespread, yet selective, protein degradation events in vitro. Cellular proteasomal activity was inhibited with APAP treatment but not with the comparatively nonhepatotoxic APAP regioisomer, N-acetyl-m-aminophenol (AMAP). Low concentrations of the proteasome-directed inhibitor MG132 (N-carbobenzoxyl-Leu-Leu-Leucinal) increased chromatin condensation and cellular stress responses preferentially in AMAP-treated cultures, suggesting a contribution of the proteasome in APAP- but not AMAP-mediated cell death. APAP-specific alterations to mitochondria were observed morphologically with evidence of mitochondrial proliferation in vitro. Biochemical alterations to cellular proteolytic events were also found in vivo, including APAP- or AMAP-mediated inhibition of caspase-3 processing. These results indicate that, although retaining some attributes of apoptosis, both APAP- and AMAP-mediated cell death have additional distinctive features consistent with longer term necrosis.

Acetaminophen↗

PubMed2002

Bcl-2 expression inhibits liver carcinogenesis and delays the development of proliferating foci.

Tumor development is thought to require both increased proliferation and inhibition of apoptosis. However, the relationship between cell replication and cell death in liver tumorigenesis is complex because both proliferation and apoptosis increase during hepatocarcinogenesis. To investigate the effect of the anti-apoptotic gene Bcl-2 in liver carcinogenesis, we established a line of double transgenic mice that express transforming growth factor-alpha (TGF-alpha), a liver mitogen, and Bcl-2. Double transgenic mice, TGF-alpha and Bcl-2 single transgenics, and wild type received an injection of diethylnitrosamine at 15 days of age. This alkylating agent induces liver carcinogenesis and its effect is greatly enhanced by TGF-alpha. We report that Bcl-2 expression inhibited diethylnitrosamine-induced liver carcinogenesis and counteracted the enhancing effect of TGF-alpha. Bcl-2 delayed the growth of proliferative foci at the early stages of carcinogenesis and inhibited cell proliferation in these foci. The effect of Bcl-2 on liver carcinogenesis is consistent with its reported ability to interfere with cell replication. The data demonstrate that the expression of an anti-apoptotic gene during liver carcinogenesis causes a delay rather than an increase in tumorigenesis.

Alkylating Agents↗