A Nuclear Protein Regulated during the Transition from Active to Quiescent Phenotype in Cultured Endothelial Cells

Abstract

Pigpen is a 67-kDa Sepharose-binding molecule isolated from mammalian endothelial and retinal pigmented epithelial cells. The protein is distributed nonhomogeneously in the nucleus, exhibiting diffuse staining throughout (excluding nucleoli), together with a small number of intensely stained focal points, or granules, and punctate staining along the nuclear envelope. Pigpen was absent or greatly attenuated in the nonepithelial cell types we examined, including fibroblasts, myeloma, and astroglia. cDNA sequence analysis revealed a positively charged molecule with an RNP-CS RNA-binding domain, 19 RGG repeats, and a consensus tyrosine phosphorylation site in the C-terminus. The amino terminal portion of the molecule is characterized by 7 glutamine-rich hexapeptide repeats similar to those found in the transactivation domain of known transcription activators. Pigpen has a high level of identity with the FUS gene product, TLS (Translocated in Liposarcoma; Crozat et al, 1993; Rabbits et al., 1993), a new member of the EWS family of proteins. Expression of pigpen is regulated during the transition between active and quiescent endothelial cell phenotypes. Both mRNA and overall protein levels are maintained at a steady level in actively growing cells. The number of nuclear granules increases as cultures approach confluency. When cells reach confluency, overall expression is sharply reduced and the number of nuclear focal points declines gradually. We observed that reactivation of endothelial cells locally by wounding of confluent cultures resulted in a spatially restricted reactivation of pigpen expression. This pattern of expression, taken together with structural data, suggests that pigpen may function in the growth and differentiation of endothelial cells during angiogenesis.

Nuclear Injection of Anti-pigpen Antibodies Inhibits Endothelial Cell Division

“…Importantly, it was recently shown that pigpen is highly expressed in rat brain tumor microvessels but is absent in mature brain vasculature, even in zones immediately adjacent to the tumor (7). Together with other observations on the up-regulation of pigpen in wounded vascular cell monolayers (5,7,8), these data suggest that pigpen's expression pattern underlies a functional role in regulating EC phenotype.We tested the hypothesis that pigpen is important for cell division by microinjection of anti-pigpen Fab fragments into the nuclei of EC in proliferating cultures. More than 200 individual cells were injected, tracked, and assayed for progression through mitosis.…”

“…Importantly, it was recently shown that pigpen is highly expressed in rat brain tumor microvessels but is absent in mature brain vasculature, even in zones immediately adjacent to the tumor (7). Together with other observations on the up-regulation of pigpen in wounded vascular cell monolayers (5,7,8), these data suggest that pigpen's expression pattern underlies a functional role in regulating EC phenotype.…”

“…This molecule displays a transcription activation domain as well as consensus motifs associated with RNA binding (5). In the latter case, we have evidence to suggest that pigpen interacts selectively with mRNAs, notably those of positive cell cycle regulators such as Bub1, E2F4, and katanin (6).…”

Insights into developmental mechanisms and cancers in the mammalian intestine derived from serial analysis of gene expression and study of the hepatoma-derived growth factor (HDGF)

“…Trp53 is commonly mutated in cancers and elevated protein levels are believed to result from post-translational stabilization (Kubbutat et al, 1997); our study reveals increased mRNA levels of this developmentally regulated gene. Tls/Fus [also called pigpen in the mouse (Alliegro and Alliegro, 1996)] is a recurring target of chromosomal translocation in human myxoid liposarcoma (Rabbitts et al, 1993), where its pathogenic mechanisms are unclear.…”

Genomic analysis of neural crest induction

“…Interestingly, 3 of the 4 RNA-binding proteins in our collection have been previously implicated in stem cell maintenance: pumilio controls germline stem cells in C. elegans and Drosophila (Crittenden et al, 2002), pigpen expression correlates with undifferentiated, proliferative endothelial cells (Alliegro and Alliegro, 1996;Alliegro, 2001), and dyskerin is a component of telomerase that appears to be essential for stem cell renewal Molecular profile of neural crest precursors in a variety of tissues including the basal layer of the epidermis (reviewed by Marcinian et al, 2000). Since the neural crest is a multipotent stem cell population, these proteins are obvious candidates for post-transcriptional maintenance of the undifferentiated, stem cell state of neural crest cells as well.…”