Eric A. Hudson

Differential expression and responsiveness of chemokine receptors (CXCR1–3) by human microvascular endothelial cells and umbilical vein endothelial cells

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)

Regulation of P311 expression by Met-hepatocyte growth factor/scatter factor and the ubiquitin/proteasome system.

P311 is a mouse cDNA originally identified for its high expression in late-stage embryonic brain and adult cerebellum, hippocampus, and olfactory bulb. The protein product of P311, however, had not been identified previously, and its function remains unknown. We report here that P311 expression is regulated at multiple levels by pathways that control cellular transformation. P311 mRNA expression was decreased sharply in both neural and smooth muscle cells when the cells were transformed by coexpression of the oncogenic tyrosine kinase receptor Met and its ligand hepatocyte growth factor/scatter factor. The P311 mRNA was found to encode an 8-kDa polypeptide that was subject to rapid degradation by the lactacystin-sensitive ubiquitin/proteasome system and an unidentified metalloprotease, resulting in a protein half-life of about 5 min. These data suggest that P311 expression is dramatically decreased by several pathways that regulate cellular growth.

Endocytosis of gastrin in cancer cells expressing gastrin/CCK-B receptor

Abstract.Endocytosis of gastrin was studied in a number of gastrin-receptor-expressing cell lines by confocal laser scanning microscopy (CLSM) with the aid of a biologically active fluorescent derivative, rhodamine green heptagastrin. Rapid clustering (within 4–7 min) and internalization of fluorescent ligand upon binding at room temperature and 37° C were observed in the rat pancreatic acinar carcinoma cell line AR42J, human gastric carcinomas AGS-P and SIIA, human colon carcinomas HCT116 and HT29, and in NIH/3T3 cells transfected with human and rat gastrin/cholecystokinin-B receptor cDNA. Internalization was inhibited by hypertonic medium. Fluorescent heptagastrin and transferrin colocalized in the same endocytic vesicles at different stages of internalization suggesting that endocytosis occurred predominantly through a clathrin-dependent mechanism. At 37° C partial colocalization with the lysosomal marker neutral red was detected by CLSM, implying that internalized gastrin accumulated in the lysosomes. Immunoelectron microscopy studies with antibodies against gastrin revealed the presence of the internalized hormone in multivesicular vesicles and endosomes. Almost no hormone was detected in lysosomes with the antibodies to gastrin, suggesting that the degradation of the peptide is rapid in those vesicles. Continuous accumulation of fluorescent label was observed by CLSM in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the gastrin receptor is recycled back to the cell membrane after hormone delivery to intracellular compartments. An estimated average recycling time for the receptor molecules was 1 h in NIH/3T3 cells.

Synthesis and properties of three fluorescent derivatives of gastrin

As a first step in the study of hormone interaction with gastrin receptor-expressing cells, three fluorescent derivatives of heptagastrin were synthesized, characterized and tested for specificity and affinity towards gastrin/CCKB receptor by means of confocal laser scanning microscopy (CLSM). Cyanine dye Cy3.29 and borfluoropyrromethene (BODIPY) derivatives of the hormone were found to be absorbed into the cells and concentrated in perinuclear organelles by a non-receptor mediated process. The BODIPY derivative turned out to be chemically unstable and was bleached by the laser beam very rapidly. Rhodamine Green-heptagastrin retained a high affinity toward the gastrin receptor (Kd=45 nm in displacement of 125I-labeled cholecystokinin-8) and showed specific binding to NIH/3T3 cells stably transfected with human gastrin/CCKB receptor cDNA, but not to nontransfected 3T3 cells. The fluorescent signal of all three dyes was sufficiently intense for localization of the compounds in cells by means of CLSM. Rhodamine Green derivative was found to be a useful tool for the study of endocytosis of the hormone. It can also be utilized for quantitative estimation of binding and determination of Kd instead of the traditionally used radiolabeled derivatives of gastrin.

Anti-peptide antibodies specific for the gastrin/cholecystokinin-B receptor

Seven synthetic peptides, between 7–22 residues long, corresponding to six different parts of the gastrin/CCKB receptor molecule which are conserved among the species, were used for raising antibodies. The peptides were coupled to keyhole limpet hemocyanine and injected into rabbits. ELISA analysis demonstrated that all peptides produced an immune response after three to six injections given at biweekly intervals. The titer ranged from 1:104 to 1:105. All antibodies recognized a 78 kDa protein on immunoblots of NIH 3T3 cells stably transfected with human gastrin/CCKB receptor cDNA, as well as human and guinea pig stomach mucosal extracts. Preincubation of the sera with the corresponding peptides abolished the staining. Indirect immunofluorescence staining revealed that four antibodies out of the seven tested recognized the receptor in fixed COS-7 cells transiently transfected with human gastrin/CCKB receptor cDNA. The reactive antibodies were raised against the peptides corresponding to receptor residues 40–58, 153–160, 288–294 and 356–372. Immunohistochemical staining of guinea pig stomach using these antisera resulted in intense staining of parietal cells in the fundus and cardia regions.