Tks5/Fish is a scaffolding protein with five SH3 domains and one PX domain. In Src-transformed cells, Tks5/Fish localizes to podosomes, discrete protrusions of the ventral membrane. We generated ...Src-transformed cells with reduced Tks5/Fish levels. They no longer formed podosomes, did not degrade gelatin, and were poorly invasive. We detected Tks5/Fish expression in podosomes in invasive cancer cells, as well as in human breast cancer and melanoma samples. Tks5/Fish expression was also required for protease-driven matrigel invasion in human cancer cells. Finally, coexpression of Tks5/Fish and Src in epithelial cells resulted in the appearance of podosomes. Thus, Tks5/Fish appears to be required for podosome formation, for degradation of the extracellular matrix, and for invasion of some cancer cells.
Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the ...related gene Lrp6. We found that compound mutants had dose‐dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development.
Introduction: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co‐receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed.
Materials and Methods: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and μCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture.
Results: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6+/−;Lrp5+/− or Lrp6+/−;Lrp5−/−) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6+/−;Lrp5−/− mice were more severely affected than Lrp6+/+;Lrp5−/− mice, whereas Lrp6+/−;Lrp5+/− mice had statistically higher BMD than Lrp6+/+;Lrp5−/− mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone.
Conclusions: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.
The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the ...mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.
ABSTRACT
The basis for the angiogenic effects of CXC chemokines such as interleukin 8 (IL‐8) and for angiostatic chemokines such as interferon‐inducible protein 10 (IP‐10) has been difficult to ...assess. We recently reported, based on an RNase protection assay, that human umbilical vein endothelial cells (HUVECs) did not express detectable mRNA for the IL‐8 receptors CXCR1 and CXCR2. This raised the possibility of heterogeneity of receptor expression by different endothelial cell (ECs) types. Since systemic angiogenesis induced by IL‐8 would more likely involve microvessel ECs, we investigated CXC receptor expression on human microvascular dermal endothelial cells (HMECs). By confocal microscopy and immunofluorescence we observed that HMECs consistently expressed high levels of CXCR1 and CXCR4 (mean fluorescence intensity of 261 ±22.1 and 306.2±19, respectively) and intermediate levels of CXCR3 and CXCR2 (173.9±30.2 and 156±30.9, respectively). In contrast, only a small proportion of HUVEC preparations expressed low levels of CXCR1, ‐2, and ‐3 (66±19.9; 49±15, and 81.4±17.9, respectively). However, both HMECs and HUVECs expressed equal levels of CXCR4. As expected, HMECs had more potent chemotactic responses to IL‐8 than HUVECs, and this was correlated with the levels of IL‐8 receptors on the ECs. Antibodies to CXCR1 and CXCR2 each had inhibitory effects on chemotaxis of HMECs to IL‐8, indicating that both IL‐8 receptors contributed to the migratory response of these cells toward IL‐8. Assessment of the functional capacity of CXCR3 unexpectedly revealed that HMECs migrated in response to relatively higher concentrations (100–500 ng/ml) of each of the ‘angiostatic’ chemokines IP‐10, ITAC, and MIG. Despite this, the ‘angiostatic’ chemokines inhibited the chemotactic response of HMECs to IL‐8. IL‐8 and SDF‐1α but not IP‐10 induced calcium mobilization in adherent ECs, suggesting that signaling events associated with calcium mobilization are separable from those required for chemotaxis. Taken together, our data indicated that functional differences among EC types is dependent on the level of the expression of CXC chemokine receptors. Whether this heterogeneityin receptor expression by ECs reflects distinct differentiation pathways remains to be established.—Salcedo, R., Resau, J. H., Halverson, D., Hudson, E. A., Dambach, M., Powell, D., Wasserman, K., Oppenheim, J. J. Differential expression and responsiveness of chemokine receptors (CXCR1–3) by human microvascular endothelial cells and umbilical vein endothelial cells. FASEB J. 14, 2055–2064 (2000)
Using double immunofluorescence staining and quantitative confocal laser scan microscopy, we show that the intensity of hepatocyte growth factor/scatter factor (HGF/SF) and Met staining in human ...primary brain tumors increases with the grade of malignancy and is prevalent in both the infiltrating tumor cells and endothelial hyperplastic areas. HGF/SF and Met also are expressed in vitro in glioblastoma multiforme cell lines as well as in normal human astrocyte (NHA) cells. Moreover, HGF/SF stimulates tyrosine phosphorylation of Met in both glioma cell lines and NHA cells, but only the glioma cell lines proliferate and become motile and invasive in response to HGF/SF, whereas the NHA cells are nonresponsive. These results implicate autocrine/paracrine Met-HGF/SF signaling in glioma tumorigenesis and suggest that HGF/SF signaling through Met is negatively regulated in NHA cells.
Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the ...phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry. These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance. High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan. Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression. Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells. Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein. Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance. Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required.
The c-KIT proto-oncogene has been implicated in the pathogenesis of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and mast cell tumors (MCTs) in ...canines. Cutaneous MCTs are common neoplasms in dogs and have a variable biologic behavior. The goal of this study was to define the prognostic significance of c-KIT mutations identified in canine MCTs and the associations between c-KIT mutations, KIT localization, and KIT expression levels. Microdissection and polymerase chain reaction were performed on 60 MCTs to identify c-KIT mutations. Anti-KIT antibodies were used for immunohistochemical evaluation of KIT localization. Forty-two MCTs were included in a tissue microarray, and KIT expression was quantified using immunofluorescence. Canine MCTs with c-KIT mutations were significantly associated with an increased incidence of recurrent disease and death. c-KIT mutations were also significantly associated with aberrant protein localization; however, the level of KIT expression did not correlate with either c-KIT mutations or changes in protein localization. Considering the high prevalence of canine MCTs and the central role of c-KIT in the tumorigenesis of certain tumors, canine MCTs are an excellent model for characterizing the role of c-KIT in neoplastic diseases and is a potential target for novel therapeutic agents in clinical trials.
T lymphocyte activation is highlighted by the induction of interleukin-2 (IL-2) gene expression, which governs much of the early lymphocyte proliferation responses. Peroxisome proliferator-activated ...receptor- gamma (PPAR gamma ) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPAR gamma mRNA expression was found in human peripheral blood T lymphocytes, raising the possibility of PPAR gamma involvement in the regulation of T cell function. Here we show that PPAR gamma ligands, troglitazone and 15-deoxy- Delta super(12,14) prostaglandin J sub(2), but not PPAR alpha agonist Wy14643, inhibited IL-2 production and phytohemagglutinin-inducible proliferation in human peripheral blood T-cells in a dose-dependent manner. This inhibitory effect on IL-2 was restricted to the PPAR gamma 2- expressing, not the PPAR gamma -lacking, subpopulation of transfected Jurkat cells. The activated PPAR gamma physically associates with transcriptional factor NFAT regulating the IL-2 promoter, blocking NFAT DNA binding and transcriptional activity. This interaction with T-cell-specific transcription factors indicates an important immunomodulatory role for PPAR gamma in T lymphocytes and could suggest a previously unrecognized clinical potential for PPAR gamma ligands as immunotherapeutic drugs to treat T-cell-mediated diseases by targeting IL-2 gene expression.
RAS genes are mutated in approximately 30% of all human cancers. Interestingly, there exists a strong bias in favor of mutation of only one of the three major RAS genes in tumors of different ...cellular origins. NRAS mutations occur in approximately 20% of human melanomas, whereas HRAS and KRAS mutations are rare in this disease. To define the mechanism(s) responsible for this preference in melanocytes, we compared the transformation efficiencies of mutant NRAS and KRAS in immortal, non-transformed Ink4a/Arf-deficient melanocytes. NRAS mutation leads to increased cellular proliferation and is potently tumorigenic. In contrast, KRAS mutation does not enhance melanocyte proliferation and is only weakly tumorigenic on its own. Although both NRAS and KRAS activate mitogen-activated protein kinase signaling, only NRAS enhances MYC activity in these cells. Our data suggest that the activity of specific RAS isoforms is context-dependent and provide a possible explanation for the prevalence of NRAS mutations in melanoma. In addition, understanding this mechanism will have important implications for cancer therapies targeting RAS pathways.
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