A NUCLEAR-PROTEIN REGULATED DURING THE TRANSITION FROM ACTIVE TO QUIESCENT PHENOTYPE IN CULTURED ENDOTHELIAL-CELLS

Pigpen is a 67-kDa Sepharose-binding molecule isolated from mammalian endothelial and retinal pigmented epithelial cells. The protein is dis tributed nonhomogeneously in the nucleus, exhibiting diffuse staining throughout (excluding nucleoli), together with a small number of inten sely stained focal points, or granules, and punctate staining along th e nuclear envelope. Pigpen was absent or greatly attenuated in the non epithelial cell types we examined, including fibroblasts, myeloma, and astroglia. cDNA sequence analysis revealed a positively charged molec ule with an RNP-CS RNA-binding domain, 19 RGG repeats, and a consensus tyrosine phosphorylation site in the C-terminus. The amino terminal p ortion of the molecule is characterized by 7 glutamine-rich hexapeptid e repeats similar to those found in the transactivation domain of know n transcription activators. Pigpen has a high level of identity with t he PUS gene product, TLS (Translocated in Liposarcoma; Crozat st al., 1993; Rabbits st al., 1993), a new member of the EWS family of protein s. Expression of pigpen is regulated during the transition between act ive and quiescent endothelial cell phenotypes. Both mRNA and overall p rotein levels are maintained at a steady level in actively growing cel ls. The number of nuclear granules increases as cultures approach conf luency. When cells reach confluency overall expression is sharply redu ced and the number of nuclear focal points declines gradually. We obse rved that reactivation of endothelial cells locally by wounding of con fluent cultures resulted in a spatially restricted reactivation of pig pen expression. This pattern of expression, taken together with struct ural data, suggests that pigpen may function in the growth and differe ntiation of endothelial cells during angiogenesis. (C) 1996 Academic P ress, Inc.