Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells

Abstract

Vascular endothelial cells undergo profound changes upon cellular activation including expression of a spectrum of cell activation-associated genes. These changes play important roles in many physiological and pathological events. By differential screening of a cDNA library prepared from interleukin-1 alpha and tumor necrosis factor-alpha-stimulated human dermal microvascular endothelial cells, we have identified a novel cytokine-inducible gene, designated as C-193. The compiled cDNA sequence of C-193 is 1901 base pairs long and shows no significant homology with any known gene sequence. Genomic DNA analysis revealed that C-193 is encoded by a single gene, which is conserved in different mammalian species. The C-193 gene was localized to human chromosome 10 by Southern blot analysis of somatic cell hybrids. Multiple AT-rich mRNA decay elements were identified in the 3'-untranslated region. C-193 mRNA expression was rapidly and transiently induced by treatment with interleukin-1 alpha or tumor necrosis factor-alpha, reached a peak of expression about 16 h post tumor necrosis factor-alpha stimulation, and the induction of C-193 was protein synthesis independent. Lipopolysaccharide and cycloheximide were also potent inducers of C-193 mRNA. Therefore, C-193 represents a new addition to the primary response gene family. In vitro translation of C-193 yielded a 36-kDa protein product, consistent with the predicted open reading frame of 318 amino acids and a calculated molecular mass of 36 kDa for C-193 protein. The predicted protein sequence contains a basic amino acid cluster similar to a nuclear localization signal, four tandem repeats of ankyrin-like sequence, and multiple consensus protein phosphorylation sites. C-193 was engineered with a FLAG tag at its carboxyl terminus and transiently expressed in COS cells. Consistent with the presence of a putative nuclear localization signal, the C-193-FLAG protein was localized to the nucleus of transfected COS cells by indirect immunofluorescence microscopy. C-193-FLAG prepared in vitro was capable of binding DNA cellulose. These results indicate that C-193 protein may play an important role in endothelial cell activation.