Role of pancreatic stellate cells in pancreatic cancer metastasis.

Pancreatic stellate cells (PSCs) produce the stromal reaction in pancreatic cancer (PC), and their interaction with cancer cells facilitates cancer progression. This study investigated the role of human PSCs (hPSCs) in the metastatic process and tumor angiogenesis using both in vivo (orthotopic model) and in vitro (cultured PSC and PC cells) approaches. A sex mismatch study (injection of male hPSCs plus female PC cells into the pancreas of female mice) was conducted to determine whether hPSCs accompany cancer cells to metastatic sites. Metastatic nodules were examined by fluorescent in situ hybridization for the presence of the Y chromosome. Angiogenesis was assessed by i) immunostaining tumors for CD31, an endothelial cell marker; and ii) quantifying human microvascular endothelial cell (HMEC-1) tube formation in vitro on exposure to conditioned media from hPSCs. Transendothelial migration was assessed in vitro by examining the movement of fluorescently labeled hPSCs through an endothelial cell monolayer. Human PSCs i) were found in multiple metastatic sites in each mouse injected with male hPSCs plus female PC cells; ii) increased CD31 expression in primary tumors from mice injected with MiaPaCa-2 and hPSCs and stimulated tube formation by HMEC-1 in vitro; and iii) exhibited transendothelial migration that was stimulated by cancer cells. Human PSCs accompany cancer cells to metastatic sites, stimulate angiogenesis, and are able to intravasate/extravasate to and from blood vessels.

Isolation of quiescent human pancreatic stellate cells: a promising in vitro tool for studies of human pancreatic stellate cell biology.

BACKGROUND: Pancreatic stellate cells (PSCs) play a critical role in pancreatic fibrosis. To date, human PSC biology has been studied using cancer- or inflammation-associated (pre-activated) PSCs, but an in vitro model of quiescent normal human PSCs (NhPSCs) has been lacking. AIMS: To (i) isolate and characterize quiescent NhPSCs, and (ii) evaluate the response of culture-activated NhPSCs to cytokines and LPS. METHODS: Quiescent NhPSCs were isolated from normal pancreatic tissue using density gradient centrifugation. PSC markers, glial fibrillary acidic protein (GFAP), desmin, α-smooth muscle actin (αSMA) and the lipopolysaccharide (LPS) receptors TLR4 and CD14 were identified by immunoblotting and immunocytochemistry. The effect of platelet-derived growth factor (PDGF), transforming growth factor ß (TGFß) and LPS on NhPSC activation was also assessed. RESULTS: Freshly isolated NhPSCs displayed a polygonal appearance with refringent cytoplasmic lipid droplets. Culture-activated NhPSCs expressed GFAP, desmin, αSMA, TLR4 and CD14, and were responsive to PDGF, TGFß and LPS. CONCLUSION: Isolated NhPSCs expressed the same markers as rat PSCs and human cancer-associated PSCs and responded to PDGF and TGFß similarly to rat PSCs. NhPSC preparations provide a useful in vitro tool to study the biology of PSCs in their physiological, non-activated state. and IAP.

Regulation of the rat CYP4A2 gene promoter by c-Jun and octamer binding protein-1.

The physiologically important cytochrome P450 (CYP) 4A2 arachidonic acid omega-hydroxylase gene is widely expressed in rat tissues. Although the induction of CYPs 4A by peroxisome proliferators and dietary lipids is established there is minimal information on the factors that control constitutive expression. To address this issue we cloned 1.4 kb of the CYP4A2 5'-upstream region and identified several DNA elements that resembled the activator protein-1 (AP-1) consensus sequence. Using a series of 5'-truncated reporter constructs a 42 bp region was detected that was responsive to the AP-1 factor c-Jun, which is important in basal gene regulation. The roles of two putative AP-1 elements at -47/-41 and -31/-25 were tested, with the former emerging from studies with mutagenised constructs as the functionally important site. These findings were supported by electromobility shift assay (EMSA) studies that indicated the interaction of the -47/-41 element with c-Jun. The -31/-25 element mediated the suppression of CYP4A2 transactivation by octamer binding protein-1 (oct-1). Thus, mutagenesis of this element relieved the modulatory effect of oct-1 on c-Jun-mediated transactivation. In EMSAs, the binding of nuclear proteins to the -31/-25 element was competed by an oct-1 consensus sequence and supershifted by an anti-oct-1 antibody. Overexpression of c-Jun in rat liver-derived H4IIE cells increased CYP4A2 mRNA to approximately 2-fold of control, but oct-1 overexpression was without significant effect. From chromatin immunoprecipitation assays both c-Jun and oct-1 bound to the CYP4A2 5'-upstream sequence in H4IIE cells. These findings implicate c-Jun and oct-1 as potentially important constitutive factors that modulate the transactivation of the CYP4A2 gene promoter.

Phospho-STAT5 accumulation in nuclear fractions from vitamin A-deficient rat liver.

The growth hormone (GH)-responsive cytochrome P450 (CYP) 2C11 is down-regulated in vitamin A-deficient (VAD) rat liver. This study assessed the impact of a VAD diet on the hepatic Janus kinase-Signal Transducers and Activators of Transcription (JAK-STAT) system that mediates GH signalling. Nuclear tyrosine- and serine-phosphorylated STAT5 accumulated in VAD liver, whereas nuclear JAK2 tyrosine kinase and SHP-1 phosphatase were decreased. Tyrosine-phosphorylated SHP-1 was decreased to 36+/-14% of control (P<0.01), indicating its impaired activation in VAD liver. Episodic GH pulses increased nuclear phospho-STAT5, especially in control liver, but nuclear phospho-JAK2 and phospho-SHP-1 were not restored. CYP2C11 protein and testosterone 16alpha-hydroxylation were decreased in VAD liver to 67+/-16% and 76+/-19% of control, and were further decreased by GH to 32+/-8% and 30+/-14% of control. Thus, hypo-responsiveness of JAK-STAT in VAD liver is associated with impaired nuclear phospho-STAT dephosphorylation.

Pretranslational upregulation of microsomal CYP4A in rat liver by intake of a high-sucrose, lipid-devoid diet containing orotic acid.

In rodents, high-fat diets promote hepatic lipid accumulation in rodents, activation of peroxisome proliferator activated receptor-alpha (PPARalpha) and upregulation of cytochrome P450 (CYP) 4A gene expression. Lipid-devoid diets containing sucrose and orotic acid (S/OA-diet) also cause lipid infiltration by stimulating intrahepatic lipid synthesis and preventing lipoprotein transport through the Golgi apparatus. This study evaluated the impact of the lipid-deficient S/OA-diet on CYP4A expression and PPARalpha activation in rodent liver. CYP4A protein and laurate omega-hydroxylation activity were increased in rat liver after S/OA-feeding for 21 days. CYP4A1 and CYP4A2 mRNAs were induced to 2.1- and 2.6-fold of control, but mRNAs corresponding to CYP4A3 and the peroxisomal acyl-CoA oxidase (AOX) were unchanged. Coadministration of clofibric acid and the S/OA-diet prevented hepatic lipid accumulation and upregulated CYP4A protein to levels comparable with clofibric acid alone (five-fold of control). Clofibric acid, alone and in combination with the S/OA-diet, upregulated CYP4A1-3 and AOX mRNAs. Hepatic PPARalpha protein was decreased by the S/OA-diet but was increased to 5.7-fold of control by clofibric acid; retinoid X-receptor-alpha (RXRalpha) protein was decreased to 26-41% of control by all treatments. In further studies, administration of the S/OA-diet to control and PPARalpha-null mice promoted hepatic lipid deposition; microsomal CYP4A protein was induced in wild-type but not PPARalpha-null mice. These findings implicate PPARalpha in the induction of CYP4A in rodent liver by the lipid-devoid S/OA-diet. Decreased availability of hepatic PPARalpha and RXRalpha after intake of the diet may contribute to the selective upregulation of hepatic CYP4A1 and CYP4A2 in this model.

Upregulation of cytochromes P450 2B in rat liver by orphenadrine.

1 The alkylamine drug orphenadrine (ORPH) is an inducer and inhibitor of the microsomal cytochrome P450 (CYP) system in mammals. This study evaluated the selectivity of CYP induction by ORPH in rat liver. 2 Immunoblot analysis indicated that ORPH was a selective inducer of the phenobarbitone (PB)-inducible CYP2B in rat liver. CYP2B protein was increased to approximately 14-fold of levels in untreated rat liver. By comparison PB increased CYP2B expression 40-fold. Corresponding increases in the activity of CYP2B-dependent androstenedione 16beta-hydroxylation were measured in microsomes from ORPH and PB-induced rats. 3 Northern analysis indicated that CYP2B1/2 mRNA was increased in ORPH-induced rat liver. Consistent with this finding, ORPH was found to activate a PB-responsive enhancer module in constitutive androstane receptor (CAR)-transfected Hep G2 cells. 4 Other alkylamines like troleandomycin impair CYP turnover. We tested whether ORPH induction of CYP2B may include a post-translational component. In PB-pretreated animals ORPH administration delayed the loss of CYP2B after PB withdrawal, but no evidence for altered turnover was found. 5 These studies establish ORPH as a selective inducer of CYP2B in rat liver. Induction appears to be mediated pretranslationally by CAR activation of CYP2B gene transcription. Post-translational stabilisation by an ORPH metabolite does not elicit induction. Induction of CYP2B may influence pharmacokinetic interactions involving ORPH.

Role of activator protein-1 in the down-regulation of the human CYP2J2 gene in hypoxia.

The cytochrome P450 (CYP) 2J2 arachidonic acid epoxygenase gene was down-regulated at a pre-translational level in human hepatoma-derived HepG2 cells incubated in a hypoxic environment; under these conditions, the expression of c-Jun and c-Fos mRNA and protein was increased. The 5'-upstream region of the CYP2J2 gene was isolated by amplification of a 2341 bp fragment and putative regulatory elements that resembled activator protein-1 (AP-1)-like sequences were identified. From transient transfection analysis, c-Jun was found to strongly activate a CYP2J2 -luciferase reporter construct, but co-transfection with plasmids encoding c-Fos or c-Fos-related antigens, Fra-1 and -2, abrogated reporter activity. Using a series of deletion-reporter constructs, a c-Jun-responsive module was identified between bp -152 and -50 in CYP2J2 : this region contained an AP-1-like element between bp -56 and -63. The capacity of this element to interact directly with c-Jun, but not c-Fos, was confirmed by electromobility-shift assay analysis. Mutagenesis of the -56/-63 element abolished most, but not all, of the activation of CYP2J2 by c-Jun, thus implicating an additional site within the c-Jun-responsive region. The present results establish an important role for c-Jun in the control of CYP2J2 expression in liver cells. Activation of c-Fos expression by hypoxia promotes the formation of c-Jun/c-Fos heterodimers, which decrease the binding of c-Jun to the CYP2J2 upstream region, leading to gene down-regulation.