The role of sialomucin CD164 (MGC-24v or endolyn) in prostate cancer metastasis

BACKGROUND: The chemokine stromal derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 have been demonstrated to be crucial for the homing of stem cells and prostate cancers to the marrow. While screening prostate cancers for CXCL12-responsive adhesion molecules, we identified CD164 (MGC-24) as a potential regulator of homing. CD164 is known to function as a receptor that regulates stem cell localization to the bone marrow. RESULTS: Using prostate cancer cell lines, it was demonstrated that CXCL12 induced both the expression of CD164 mRNA and protein. Functional studies demonstrated that blocking CD164 on prostate cancer cell lines reduced the ability of these cells to adhere to human bone marrow endothelial cells, and invade into extracellular matrices. Human tissue microarrays stained for CD164 demonstrated a positive correlation with prostate-specific antigen levels, while its expression was negatively correlated with the expression of androgen receptor. CONCLUSION: Our findings suggest that CD164 may participate in the localization of prostate cancer cells to the marrow and is further evidence that tumor metastasis and hematopoietic stem cell trafficking may involve similar processes

VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

Angiogenesis is the process by which new blood vessels arise from existing ones by the budding out of endothelial cell capillaries from the luminal side of blood vessels. Blood vessel formation is essential for organ development during embryogenesis and is associated with several physiological and pathological processes, such as wound healing and tumor development. The VE-statin/egfl7 gene is specifically expressed in endothelial cells during embryonic development and in the adult. We studied here the regulatory mechanisms that control this tissue-specific expression. RT-qPCR analyses showed that the specificity of expression of VE-statin/egfl7 in endothelial cells is not shared with its closest neighbor genes notch1 and agpat2 on the mouse chromosome 2. Chromatin-immunoprecipitation analysis of histone modifications at the VE-statin/egfl7 locus showed that the chromatin is specifically opened in endothelial cells, but not in fibroblasts at the transcription start sites. A 13 kb genomic fragment of promoter was cloned and analyzed by gene reporter assays which showed that two conserved regions are important for the specific expression of VE-statin/egfl7 in endothelial cells; a −8409/−7563 enhancer and the −252/+38 region encompassing the exon-1b transcription start site. The latter contains essential GATA and ETS-binding sites, as assessed by linker-scanning analysis and site-directed mutagenesis. An analysis of expression of the ETS and GATA transcription factors showed that Erg, Fli-1 and GATA-2 are the most highly expressed factors in endothelial cells. Erg and GATA-2 directly control the expression of the endogenous VE-statin/egfl7 while Fli-1 probably exerts an indirect control, as assessed by RNA interference and chromatin immunoprecipitation. This first detailed analysis of the mechanisms that govern the expression of the VE-statin/egfl7 gene in endothelial cells pinpoints the specific importance of ETS and GATA factors in the specific regulation of genes in this cell lineage

The effect of overexpression of amyloid precursor protein in different cell types of vascular tissue

Vascular damage as a result of amyloid precursor protein (APP) and amyloid protein (Aβ) accumulation is observed in brain vasculature of patients with Alzheimer\u27s disease and cerebral amyloid angiopathy (CAA). To determine whether the endogenous overexpression of APP can also result in vascular cell damage, plasmids containing wild type APP cDNA or a Dutch mutant variant (associated with CAA) under the control of the CMV promoter were stably introduced into bovine aortic endothelial cells and bovine smooth muscle cells. Approximately 10 days post transfection a significant morphological change was observed in endothelial cells expressing either wild type or mutant APP cDNA. These cells exhibited a large number of vacuoles followed by gradual cell death. The cell viability assay confirmed these observations and demonstrated the toxic effect of the amyloid expression in endothelial cells. We observed no toxicity of endothelial cells transfected with a control plasmid containing the β-globin gene. The APP expression in endothelial cells was accompanied by an increase in generation of free radicals that was detected by using fluorescent dye (DCFH-DA). In striking contrast the expression of the wild type or mutant APP in smooth muscle cells had no observed toxic effects, even when cells were maintained in culture as long as 3 months post transfection. Similar expression of the APP proteins in both transfected endothelial and smooth muscle cells was confirmed by immunohistochemistry. Thus the endothelial and smooth muscle cell components of the vasculature differentially respond to an endogenous overexpression of amyloid precursor protein

Endothelial cell dysfunction in response to intracellular overexpression of amyloid precursor protein

Vascular damage as a result of amyloid protein (Aβ) accumulation is observed in brain vasculature of patients with Alzheimer\u27s disease and cerebral amyloid angiopathy (CAA). Aβ is a proteolytic product of amyloid precursor protein (APP) which is expressed in many cell types but it is not clear which cell type is the source of toxic Aβ production. Exposure of vascular endothelial and smooth muscle cells to exogenous Aβ results in toxicity in both cell types. To determine the effect of endogenous overexpression of APP in endothelial and smooth muscle cells plasmids containing wild type APP cDNA or a Dutch mutant variant (associated with CAA) were stably introduced into bovine aortic endothelial (BAE) cells and bovine smooth muscle (BSM) cells. A significant morphological change was observed in endothelial cells expressing either wild type or mutant APP cDNA These cells exhibited a large number of vacuoles followed by gradual cell death. Determination of eel! viability (MTT assay) confirmed these observations and demonstrated the toxic effect of the amyloid expression in endothelial cells. Intracellular coexpression of plasmids containing free radical scavengers cDNA (either metallothionein or manganese Superoxide dismutase), abolished the toxic effect of APP in endothelial cells. These results suggest that APP overexpression exerts its toxic effect on endothelial cells via generation of free radicals. In striking contrast the expression of the wild type or mutant APP in smooth muscle cells had no observed toxic effects, even when cells were maintained in culture as long as 3 months post transfection. Similar expression levels of the APP proteins in both transfected endothelial and smooth muscle cells was confirmed by immunohistochemistry. Thus the endothelial and smooth muscle cell components of the vasculature differentially respond to an endogenous overexpression of APP, and endothelial cells are specifically susceptible to increased endogenous APP levels

Structure and expression of the human metallothionein-IG gene. Differential promoter activity of two linked metallothionein-I genes in response to heavy metals

The human metallothionein (MT)-IG gene (hMT-IG) is tandemly linked in a head-to-head fashion with the hMT-IF gene. The hMT-IG gene encodes a MT-I polypeptide and has a tripartite structure. The 5'-flanking region of the hMT-IG gene has a TATAA box, four GC motifs, and at least four metal responsive elements. The 3'-untranslated region has a variation of the polyadenylation signal, AATTAA, and the 3'-flanking region a YGTGTTYY RNA processing signal. This gene is expressed in hepatoma-derived cell lines (Hep G2 and Hep3B2) in response to the heavy metals (cadmium, copper, and zinc) but not to the glucocorticoid analogue dexamethasone. In contrast, the lymphoblastoid cell line (Wi-L2) does not express the hMT-IG gene. These results suggest that the hMT-IG gene is regulated differentially and in a cell type-specific manner. Transient expression studies of the chloramphenicol acetyltransferase (CAT) gene under the transcriptional control of either the hMT-IG or hMT-IF promoter in Hep G2 cells has demonstrated that both promoters contain all the necessary cis-acting elements to elicit a similar pattern of heavy metal inducibility. However, the hMT-IG promoter in all instances is five times more active than the hMT-IF promoter. The differences in promoter activity of these genes could possibly be due to inherent differences in their basal level regulatory sequences. The expression of MT-IGcat in transfected Wi-L2 cells demonstrates that the hMT-IG promoter is not cell type-specific

Inosine incorporation in GC rich RNA probes increases hybridization sequence specificity

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