Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation

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Abstract

To study the changes in gene expression in endothelial cells stimulated by lipopolysaccharide (LPS) we performed subtraction hybridization on control human umbilical vein endothelial cells (HUVEC) versus HUVEC stimulated by LPS. A novel cDNA, named endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), was cloned from our differentially expressed EST database of HUVEC cDNA library (GenBank Accession No. AY074889). Computational analysis showed that EOLA1 is 1404 bp long, encoding a 158 aa, 17.8 kDa protein, mapped to chromosome Xq27.4 with 5 exons, expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. Stable transfection of EOLA1 stimulates ECV304 cell proliferation. Our data suggest that the physical interaction of EOLA1 and MT2A may have an important role of cell protection in inflammation reaction.

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Materials and methods

3and 5rapid amplification of cDNA ends. For the amplification and cloning of full-length cDNAs from the subtracted cDNA fragments, the SMART RACE cDNA amplification kit (Clontech) was used. Total RNA from ECV304 cells stimulated with LPS was isolated as described elsewhere [9]. One microgram of RNA was reverse transcribed with Powerscript reverse transcriptase according to the manufacturer’s instructions. 3′-Rapid amplification of cDNA ends (RACE) was done using the supplied universal

EOLA1 cDNA cloning and sequence analysis

After SSH, one of the six clones without homology to known genes, designated ST55 (GenBank Accession No. BM121646), demonstrated an increased expression in HUVEC stimulated by LPS in Northern blot (figure not shown). RACE of ST55 clone resulted in a full-length cDNA and this gene was called EOLA1. The cDNA of this novel gene consists of 1404 nucleotides including a 542 nucleotide 5′ untranscription region (UTR), 474 nucleotides of an ORF predicting 158 amino acids with a molecular mass of 17.89 

Acknowledgments

This work was supported by China National Natural Science Foundation of Grant (No. 30200101 and No. 30371468) and by an operating grant from China State Key Laboratory of Trauma, Burn and Combined Injury.

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Abbreviations: LPS, lipopolysaccharide; EOLA1, endothelial-overexpressed lipopolysaccharide-associated factor 1; MT2A, metallothionein 2A; HUVEC, human umbilical vein endothelial cells; SSH, suppression subtractive hybridization; LBP, LPS-binding protein; RACE, rapid amplification of cDNA ends.

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