Stromal cells mediate retinoid-dependent functions essential for renal development.
The essential role of vitamin A and its metabolites, retinoids, in kidney development has been demonstrated in vitamin A deficiency and gene targeting studies. Retinoids signal via nuclear transcription factors belonging to the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families. Inactivation of RARaplpha and RARbeta2 receptors together, but not singly, resulted in renal malformations, suggesting that within a given renal cell type, their concerted function is required for renal morphogenesis. At birth, RARalpha beta2(-) mutants displayed small kidneys, containing few ureteric bud branches, reduced numbers of nephrons and lacking the nephrogenic zone where new nephrons are continuously added. These observations have prompted us to investigate the role of RARalpha and RARbeta2 in renal development in detail. We have found that within the embryonic kidney, RARalpha and RARbeta2 are colocalized in stromal cells, but not in other renal cell types, suggesting that stromal cells mediate retinoid-dependent functions essential for renal development. Analysis of RARalpha beta2(-) mutant kidneys at embryonic stages revealed that nephrons were formed and revealed no changes in the intensity or distribution of molecular markers specific for different metanephric mesenchymal cell types. In contrast the development of the collecting duct system was greatly impaired in RARalpha beta2(-) mutant kidneys. Fewer ureteric bud branches were present, and ureteric bud ends were positioned abnormally, at a distance from the renal capsule. Analysis of genes important for ureteric bud morphogenesis revealed that the proto-oncogene c-ret was downregulated. Our results suggest that RARalpha and RARbeta2 are required for generating stromal cell signals that maintain c-ret expression in the embryonic kidney. Since c-ret signaling is required for ureteric bud morphogenesis, loss of c-ret expression is a likely cause of impaired ureteric bud branching in RARalpha beta2(-) mutants. (+info)
VEGF is required for growth and survival in neonatal mice.
We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP- mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development. (+info)
Expression of the naturally occurring truncated trkB neurotrophin receptor induces outgrowth of filopodia and processes in neuroblastoma cells.
We have investigated the effects of the truncated trkB receptor isoform T1 (trkB.T1) by transient transfection into mouse N2a neuroblastoma cells. We observed that expression of trkB.T1 leads to a striking change in cell morphology characterized by outgrowth of filopodia and processes. A similar morphological response was also observed in SH-SY5Y human neuroblastoma cells and NIH3T3 fibroblasts transfected with trkB.T1. N2a cells lack endogenous expression of trkB isoforms, but express barely detectable amounts of its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). The morphological change was ligand-independent, since addition of exogenous BDNF or NT-4 or blockade of endogenous trkB ligands did not influence this response. Filopodia and process outgrowth was significantly suppressed when full-length trkB.TK+ was cotransfected together with trkB.T1 and this inhibitory effect was blocked by tyrosine kinase inhibitor K252a. Transfection of trkB.T1 deletion mutants showed that the morphological response is dependent on the extracellular, but not the intracellular domain of the receptor. Our results suggest a novel ligand-independent role for truncated trkB in the regulation of cellular morphology. (+info)
Growth inhibition of breast cancer cells by Grb2 downregulation is correlated with inactivation of mitogen-activated protein kinase in EGFR, but not in ErbB2, cells.
Increased breast cancer growth has been associated with increased expression of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases (RTKs). Upon activation, RTKs may transmit their oncogenic signals by binding to the growth factor receptor bound protein-2 (Grb2), which in turn binds to SOS and activates the Ras/Raf/MEK/mitogen-activated protein (MAP) kinase pathway. Grb2 is important for the transformation of fibroblasts by EGFR and ErbB2; however, whether Grb2 is also important for the proliferation of breast cancer cells expressing these RTKs is unclear. We have used liposomes to deliver nuclease-resistant antisense oligodeoxynucleotides (oligos) specific for the GRB2 mRNA to breast cancer cells. Grb2 protein downregulation could inhibit breast cancer cell growth; the degree of growth inhibition was dependent upon the activation and/or endogenous levels of the RTKs. Grb2 inhibition led to MAP kinase inactivation in EGFR, but not in ErbB2, breast cancer cells, suggesting that different pathways might be used by EGFR and ErbB2 to regulate breast cancer growth. (+info)
Over-representation of a germline RET sequence variant in patients with sporadic medullary thyroid carcinoma and somatic RET codon 918 mutation.
The aetiology of sporadic medullary thyroid carcinoma is unknown. About 50% harbour a somatic mutation at codon 918 of RET (M918T). To investigate whether other RET sequence variants may be associated with or predispose to the development of sporadic medullary thyroid carcinoma, we analysed genomic DNA from the germline and corresponding tumour from 50 patients to identify RET sequence variants. In one patient, tumour DNA showed a novel somatic 12 bp in-frame deletion in exon 15. More interestingly, we found that the rare polymorphism at codon 836 (c.2439C > T; S836S) occurred at a significantly higher frequency than that in control individuals without sporadic medullary thyroid carcinoma (Fisher's exact test, P = 0.03). Further, among the nine evaluable cases with germline c.2439C/T, eight also had the somatic M918T mutation in MTC DNA which was more frequent than in patients with the more common c.2439C/C (89% vs 40%, respectively; Fisher's exact test, P = 0.01). These findings suggest that the rare sequence variant at codon 836 may somehow play a role in the genesis of sporadic medullary thyroid carcinoma. (+info)
Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2.
Interaction between integrin alphavbeta3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin-mediated outside-in signals co-operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between alphavbeta3 integrin and tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) in human endothelial cells. We report that tyrosine-phosphorylated VEGFR-2 co-immunoprecipitated with beta3 integrin subunit, but not with beta1 or beta5, from cells stimulated with VEGF-A165. VEGFR-2 phosphorylation and mitogenicity induced by VEGF-A165 were enhanced in cells plated on the alphavbeta3 ligand, vitronectin, compared with cells plated on the alpha5beta1 ligand, fibronectin or the alpha2beta1 ligand, collagen. BV4 anti-beta3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR-2; (ii) the activation of downstream transductor phosphoinositide 3-OH kinase; and (iii) biological effects triggered by VEGF-A165. These results indicate a new role for alphavbeta3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, alphavbeta3 integrin participates in the full activation of VEGFR-2 triggered by VEGF-A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration. (+info)
Socs1 binds to multiple signalling proteins and suppresses steel factor-dependent proliferation.
We have identified Socs1 as a downstream component of the Kit receptor tyrosine kinase signalling pathway. We show that the expression of Socs1 mRNA is rapidly increased in primary bone marrow-derived mast cells following exposure to Steel factor, and Socs1 inducibly binds to the Kit receptor tyrosine kinase via its Src homology 2 (SH2) domain. Previous studies have shown that Socs1 suppresses cytokine-mediated differentiation in M1 cells inhibiting Janus family kinases. In contrast, constitutive expression of Socs1 suppresses the mitogenic potential of Kit while maintaining Steel factor-dependent cell survival signals. Unlike Janus kinases, Socs1 does not inhibit the catalytic activity of the Kit tyrosine kinase. In order to define the mechanism by which Socs1-mediated suppression of Kit-dependent mitogenesis occurs, we demonstrate that Socs1 binds to the signalling proteins Grb-2 and the Rho-family guanine nucleotide exchange factors Vav. We show that Grb2 binds Socs1 via its SH3 domains to putative diproline determinants located in the N-terminus of Socs1, and Socs1 binds to the N-terminal regulatory region of Vav. These data suggest that Socs1 is an inducible switch which modulates proliferative signals in favour of cell survival signals and functions as an adaptor protein in receptor tyrosine kinase signalling pathways. (+info)
Control of growth and differentiation by Drosophila RasGAP, a homolog of p120 Ras-GTPase-activating protein.
Mammalian Ras GTPase-activating protein (GAP), p120 Ras-GAP, has been implicated as both a downregulator and effector of Ras proteins, but its precise role in Ras-mediated signal transduction pathways is unclear. To begin a genetic analysis of the role of p120 Ras-GAP we identified a homolog from the fruit fly Drosophila melanogaster through its ability to complement the sterility of a Schizosaccharomyces pombe (fission yeast) gap1 mutant strain. Like its mammalian homolog, Drosophila RasGAP stimulated the intrinsic GTPase activity of normal mammalian H-Ras but not that of the oncogenic Val12 mutant. RasGAP was tyrosine phosphorylated in embryos and its Src homology 2 (SH2) domains could bind in vitro to a small number of tyrosine-phosphorylated proteins expressed at various developmental stages. Ectopic expression of RasGAP in the wing imaginal disc reduced the size of the adult wing by up to 45% and suppressed ectopic wing vein formation caused by expression of activated forms of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family. The in vivo effects of RasGAP overexpression required intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for its activity. These results show that RasGAP can function as an inhibitor of signaling pathways mediated by Ras and receptor tyrosine kinases in vivo. Genetic interactions, however, suggested a Ras-independent role for RasGAP in the regulation of growth. The system described here should enable genetic screens to be performed to identify regulators and effectors of p120 Ras-GAP. (+info)