ELR+-CXC chemokines and their receptors in early metanephric development.

Although originally identified as mediators of inflammation, it is now apparent that chemokines play a fundamental role in tissue development. In this study, ELR(+)-CXC chemokine family members CXCL2 and CXCL7, along with their preferred receptor CXCR2, were expressed at the earliest stages of metanephric development in the rat, and signaling through this receptor was required for the survival and maintenance of the undifferentiated metanephric mesenchyme (MM). A specific antagonist of the CXCR2 receptor SB225002 induced apoptosis in this population but did not affect more mature structures or cells in the ureteric bud. CXCL7 treatment of isolated MM elicited an angiogenic response by upregulation of matrix metalloprotease 9 and endothelial and mesangial markers (platelet-endothelial cell adhesion molecule, Megsin, Thy-1, PDGF receptor alpha, and vascular alpha-actin) and induced SB225002-sensitive cell invasion through a matrix. Because Wilms' tumor cells may similarly depend on CXCR2 signaling for survival, primary tumor samples were analyzed, and 15 of 16 Wilms' tumors were found to be CXCR2 positive, whereas grossly normal kidney tissues from tumor patients or renal cell carcinomas were CXCR2 negative. Furthermore, cell lines derived from Wilms' tumors but not those from renal cell carcinomas were sensitive to SB225002-induced apoptosis. These data provide evidence for a prosurvival and proangiogenic role of ELR(+)-CXC chemokines and their receptor CXCR2 during metanephric development and suggest a novel mechanism for chemotherapeutic intervention in Wilms' tumor.

Species-specific effects of the hepatocarcinogens 3'-methyl-4-dimethyl-aminoazobenzene and ortho-aminoazotoluene in mouse and rat liver.

The effects of rat-specific hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), mouse-specific hepatocarcinogen ortho-aminoazotoluene (OAT), non-species-specific hepatocarcinogen diethylnitrosamine (DENA), and non-carcinogenic 4'-methyl-4-dimethylaminoazobenzene (4'-MeDAB) on glucocorticoid induction of tyrosine aminotransferase (TAT) and DNA-binding activity of hepatocyte nuclear factor 3 (HNF3) family of transcription factors were investigated with carcinogen-susceptible and -resistant animals. Species-specific hepatocarcinogens 3'-MeDAB and OAT strongly inhibited glucocorticoid induction of TAT in the liver of susceptible but not resistant animals. DENA, which is highly carcinogenic for the liver of both rats and mice inhibited glucocorticoid induction of TAT in both species, while non-carcinogenic 4'-MeDAB was absolutely ineffective both in rats and mice. The inhibition of TAT activity by the carcinogens was due to reduced levels of TAT mRNA, which is most likely to be a result of the reduced rate of transcription initiation of the TAT gene. In all cases, the TAT inhibition was accompanied by significant reduction of DNA-binding activity of the HNF3 transcription factor, which is known to be critical to glucocorticoid regulation of TAT gene. We also demonstrated that the described species-specific effects of OAT and of 3'-MeDAB on HNF3 DNA-binding activity may be initiated not only by administration in vivo, but also by their direct administration to homogenate, intact nuclei or nuclear lysate, but not to nuclear extract fraction, obtained by precipitation with 0.32 g/mL of ammonium sulfate (Fraction I). We showed, that a factor responsible for this effect might be precipitated in 0.32-0.47 g/mL interval of ammonium sulfate concentration. In contrast, non-specific hepatocarcinogen DENA was effective upon being added directly to Fraction I, implying a different mechanism of its action.

Conditionally immortalized cell line of inducible metanephric mesenchyme.

BACKGROUND: The mesenchymal-epithelial conversion of metanephric mesenchyme (MM) in the formation of nephronic tubules has long served as a paradigm for inductive signaling in morphogenesis. However, the mechanisms underlying this differentiation have remained an enigma due to insufficient numbers of primary mesenchymal cells that must be isolated manually from animal embryos. To overcome this problem, we have established a conditionally immortalized cell line, the rat-inducible metanephric mesenchyme (RIMM-18) by transfection of primary mesenchymal cells with a vector, encoding an estradiol-dependent E1A-ER fusion protein. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), luciferase reporter assay, electrophoretic mobility shift assay, immunocytochemical, and immunohistochemical stainings were used to characterize the established cell line. RESULTS: We demonstrate that in the presence of estradiol, the RIMM-18 cell line proliferates continuously, maintaining mesenchymal characteristics for over 40 passages. These cells are vimentin-positive and cytokeratin-negative. Under inductive conditions in the absence of estradiol, they are responsive to a number of cytokines, which are established inducers of mesenchymal cells in vivo and in vitro [i.e., fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and transforming growth factor-beta 2 (TGF-beta 2)]. We show the presence in RIMM-18 cells of specific protein markers and functionally active signaling pathways required for induction of tubule formation in MM. Furthermore, induced RIMM-18 cells change morphology, acquiring epithelial-like features, and begin to express epithelial markers (e.g., E-cadherin, cytokeratin, gamma-glutamyl-transpeptidase, and secreted frizzled-related protein 2 (sFRP2). CONCLUSION: This preliminary characterization of the RIMM-18 cell line suggests that it will be useful in the study of biochemical and molecular mechanisms of nephronic development and, possibly, of some types of renal cancer such as Wilms' tumor, which caricatures the normal process of kidney development.