Angiogenic endothelium-specific nestin expression is enhanced by the first intron of the nestin gene

Abstract

Nestin is a member of intermediate filaments abundantly expressed in neural stem cells and glioblastomas. The nestin gene has four exons and three introns, and neural cell-specific expression is regulated by the second intron. We previously reported that nestin was invariably detected in the tumor endothelium in gliomas even though tumor cells were negative for nestin. In the present study, we further confirmed nestin immunostaining in tumor endothelium of a variety of common cancers, including lung, stomach, colon, and cervical carcinomas. We examined an endothelium-specific regulator using human umbilical vein endothelial cells (HUVECs) and human glioblastoma-derived U251 cells. In a luciferase reporter assay, the first intron plus 5 0 upstream promoter (5 0 UP) gave the highest activity, followed by 5 0 UP, and the second intron plus 5 0 UP. However, the assay values were much lower by HUVEC extracts than by U251 cell extracts. Although green fluorescent protein expression was positive over all U251 cells under either the first intron, second intron, or ubiquitously active CAG promoter, the fluorescence in HUVECs was limited to a few cells even under the first intron. This difference came from the growth feature of HUVECs which exhibit growth arrest by contact inhibition. We found that the nestin expression was specific to proliferative endothelium, by using proliferation markers in hemangioblastomas and in situ hybridization. Using an endothelial tube formation assay, tyrosine kinase domain-deleted VEGF receptor KDR effectively abolished the tube formation under the first intron. We suggest that the nestin expression in tumor endothelium is enhanced by the first intron.

Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow

“…Endothelial nestin expression has been previously described in vivo, primarily in solid tumors and the brain, as well as in endothelial cell lines in culture. 40,41 Nestin expression within subendothelial cells within the vessel wall-internal to the SMA þ VSMP layer but external to the von Willebrand factor þ layer-has recently been reported; 42 we also identified occasional nestin þ , CD34À subendothelial cells. The relationship between these nonendothelial nestin þ vascular wall cells and vessel wall-resident mesenchymal stem or progenitor cell populations 43 is unclear.…”

The Wilms' tumour suppressor WT1 is involved in endothelial cell proliferation and migration: expression in tumour vessels in vivo

“…A widely accepted marker of neovascularization is the intermediate filament protein nestin, which was detected in the majority of tumour vessels and in newly forming vessels after myocardial infarction (Aihara et al, 2004;Mokry et al, 2004). Furthermore, mice expressing green fluorescent protein under control of nestin regulatory sequences are used to monitor formation of new vessels during tumour progression (Amoh et al, 2005).…”

“…As we could show recently that nestin, which is regarded as a marker of neovascularization (Aihara et al, 2004;Mokry et al, 2004), is expressed in the developing heart and kidney and regulated by WT1, we reasoned that nestin might act downstream of WT1 also in endothelial cells and in tumour vessels. Indeed, inhibition of WT1 expression by stealth RNAi significantly reduced nestin protein expression in endothelial cells as determined by western blotting ( Figure 5A).…”

Expression of MYCN in pediatric synovial sarcoma

“…For example, the MCM7 protein has numerous transactivation sites for the E2F transcription factor, 49 and expression of MCM7 is increased by activation of E2F in fibroblasts. 50 Furthermore, the nestin gene has several putative binding sites for the transcription factors HIF-2a and GATA within its first intron, 51 suggesting a role for multiple transcription factors in nestin expression.…”