Hepatocellular expression of glutamine synthetase: an indicator of morphogen actions as master regulators of zonation in adult liver.

Glutamine synthetase (GS) has long been known to be expressed exclusively in pericentral hepatocytes most proximal to the central veins of liver lobuli. This enzyme as well as its peculiar distribution complementary to the periportal compartment for ureogenesis plays an important role in nitrogen metabolism, particularly in homeostasis of blood levels of ammonium ions and glutamine. Despite this fact and intensive studies in vivo and in vitro, many aspects of the regulation of its activity on the protein and on the genetic level remained enigmatic. Recent experimental advances using transgenic mice and new analytic tools have revealed the fundamental role of morphogens such as wingless-type MMTV integration site family member signals (Wnt), beta-catenin, and adenomatous polyposis coli in the regulation of this particular enzyme. In addition, novel information concerning the structure of transcription factor binding sites within regulatory regions of the GS gene and their interactions with signalling pathways could be collected. In this review we focus on all aspects of the regulation of GS in the liver and demonstrate how the new findings have changed our view of the determinants of liver zonation. What appeared as a simple response of hepatocytes to blood-derived factors and local cellular interactions must now be perceived as a fundamental mechanism of adult tissue patterning by morphogens that were considered mainly as regulators of developmental processes. Though GS may be the most obvious indicator of morphogen action among many other targets, elucidation of the complex regulation of the expression of the GS gene could pave the road for a better understanding of the mechanisms involved in patterning of liver parenchyma. Based on current knowledge we propose a new concept of how morphogens, hormones and other factors may act in concert, in order to restrict gene expression to small subpopulations of one differentiated cell type, the hepatocyte, in different anatomical locations. Although many details of this regulatory network are still missing, and an era of exciting new discoveries is still about to come, it can already be envisioned that similar mechanisms may well be active in other organs contributing to the fine-tuning of organ-specific functions.

Effect of growth factors on proliferation and expression of growth factor receptors in a human lens epithelial cell line.

PURPOSE: To investigate the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and transforming growth factor beta 2 (TGFbeta2) on proliferation of a human lens epithelial cell line (HLEC-SRA 01/04); the effect of bFGF and TGFbeta2 on proliferation of human lens epithelial cells (HLECs); and the expression of bFGF, EGF, IGF-1, and TGFbeta2 receptors in an HLEC-SRA 01/04 cell line. SETTING: Department of Ophthalmology, University of Ulm, Ulm, Germany. METHODS: Both HLEC and HLEC-SRA 01/04 were treated with 1 to 50 ng/mL bFGF and TGFbeta2) Additionally, HLEC-SRA 01/04 were cultured with EGF and IGF-1 at a concentration of 1 to 50 ng/mL for 48 hours in the presence of [3H]-thymidine. In all experiments, untreated serum-free negative controls were used. (3H)-thymidine incorporation as a direct measure of lens epithelial cell proliferation was assessed by liquid scintillation counting. The expression of bFGF, EGF, IGF-1, and TGFbeta2 receptors in HLEC-SRA 01/04 were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was performed using the 2-sample t test for the means. RESULTS: Proliferation of HLECs was dose dependently induced by bFGF and TGFbeta2, showing maximum effects at 10 ng/mL (P = .0003) and at 50 ng/mL (P < .0001), respectively. Proliferation of HLEC-SRA 01/04 was also induced by bFGF, showing slight but significant effects (P < .03). Additionally, HLEC-SRA 01/04 proliferation was dose-dependently induced by EGF with a maximum effect at 5 ng/mL (P < .01), IGF-1 with a maximum effect at 5 ng/mL (P < .0001), and TGFbeta2 with a maximum effect at 10 ng/mL (P < .0001) compared with the control. The RT-PCR analysis revealed bFGF, EGF, IGF-1, and TGFbeta2 receptor expression in the HLEC-SRA 01/04 cell line. CONCLUSIONS: The data showed that bFGF and TGFbeta2 are strong mitogens for HLEC. The HLEC-SRA 01/04 cell line derived from HLEC reacted to growth factors, with cell proliferation only to a lesser extent. Such quiescence of these cells, when compared with cells in primary culture, cannot be explained by the lack of respective receptors for growth factors. Further investigation of growth factor-induced responses of both cell types will provide new insight into the proliferative processes involved in postoperative secondary cataract formation.

Octreotide inhibits growth factor-induced and basal proliferation of lens epithelial cells in vitro.

PURPOSE: To evaluate whether a synthetic analog of somatostatin, octreotide, has an inhibitory effect on lens epithelial cell (LEC) proliferation induced by basal and basic fibroblast growth factor as well as insulin-like growth factor 1. METHODS: Confluent LEC cultures were kept in serum-free defined medium containing [(3)H]-thymidine in the presence of octreotide alone at the concentration range of 10(-7) M to 10(-10) M or in combination with either basic fibroblast growth factor or insulin-like growth factor 1. Additionally, the expression of somatostatin receptors (1-5) in LECs were analyzed by reverse transcription-polymerase chain reaction. RESULTS: Octreotide decreased the proliferation of human LECs in a dose-dependent manner, exhibiting a maximal inhibitory concentration at 10(-9) M (P<.03). Moreover, octreotide (10(-9) M) potently inhibited basic fibroblast growth factor- and insulin-like growth factor 1-induced bovine lens epithelial cell proliferation (P<.0001). The respective products of all 5 subtypes of somatostatin receptors were found in human LECs and somatostatin receptor type 2 in bovine LECs. CONCLUSION: The data show that, depending on the concentration, octreotide is able to decrease proliferative responses of LECs. Moreover, the cell proliferation induced by growth factors was potently inhibited by octreotide. Therefore, octreotide could be a potential drug after cataract surgery in prevention of growth factor-dependent proliferative disorders such as posterior capsule opacification and anterior capsule contraction in diabetic patients.

The RL-ET-14 cell line mediates expression of glutamine synthetase through the upstream enhancer/promoter region.

BACKGROUND/AIMS: The expression of glutamine synthetase (GS) in the mammalian liver is confined to the hepatocytes surrounding the central vein and can be induced in cultures of periportal hepatocytes by co-cultivation with the rat-liver epithelial cell line RL-ET-14. We exploited these observations to identify the regulatory regions of the GS gene and the responsible signal-transduction pathway that mediates this effect. METHODS: Fetal hepatocytes of wild-type or GS-transgenic mice were co-cultured with RL-ET-14 cells to induce GS expression. Small-interfering RNA was employed to silence beta-catenin expression in the fetal hepatocytes prior to co-culture. RESULTS: Co-cultivation of RL-ET-14 cells with fetal mouse hepatocytes induced GS expression 4.2-fold. The expression of another pericentral enzyme, ornithine aminotransferase and a periportal enzyme, carbamoylphosphate synthetase, were not affected. Co-culture of RL-ET-14 cells with transgenic fetal mouse hepatocytes demonstrated that GS expression was induced via its upstream enhancer located at -2.5 kb and that the signal mediator required a functional beta-catenin pathway. CONCLUSIONS: The 'RL-ET-14' factor specifically induces GS expression, working via its upstream enhancer in a beta-catenin-dependent fashion.