Identification of a family of alternatively spliced mRNA species of angiopoietin-1.

Angiopoietin-1 (Ang-1) is required for developing vessels, and its absence leads to defects in vessel remodeling. Ang-1 has been identified as the ligand for the tyrosine kinase receptor Tie-2, which is expressed specifically on endothelial cells and early hematopoietic cells. In studying the role of Tie-2 and Ang-1 in megakaryocytopoiesis, 3 alternatively spliced species of Ang-1 mRNA (Ang-1.3 kb, Ang-0.9 kb, and Ang-0.7 kb) were identified in addition to the full-length Ang-1 (Ang-1.5 kb), in the megakaryocyte cell line CHRF by reverse transcription-polymerase chain reaction (RT-PCR), and then cloned and sequenced. The expression of 3 alternatively spliced isoforms of Ang-1 was confirmed by RT-PCR using specific primer pairs derived from junction sites and the 3' end of Ang-1 cDNA, and it was further demonstrated by nuclease protection assay, Northern blotting, and immunoblotting in CHRF cells. Expression of the Ang-1.3 kb isoform was also detected in human primary fibroblast cell line FS4, breast cancer cell line MDAMB-468, and CD34(+)CD41(+) cells of fetal liver and platelets. The function of the 1.5-kb, 1.3-kb, and 0.9-kb isoforms was examined. Recombinant proteins Ang-1.5 and 0.9 kb bind strongly to the recombinant Tie-2 receptor (Tie-2-Fc), whereas the 1.3-kb isoform does not. The Ang-1.3 kb isoform binds to the 1.5-kb isoform. Ang-1. 5 kb, but not the 1.3-kb and 0.9-kb isoforms, induces tyrosine phosphorylation of Tie-2 in human umbilical vein endothelial cells. These data suggest that isoforms 1.3 kb and 0.9 kb could serve as dominant negative molecules for the full-length Ang-1. The possible involvement of the newly identified Ang-1 isoforms in angiogenesis and in growth and differentiation of hematopoietic progenitor cells provides a greater complexity to these processes. (Blood. 2000;95:1993-1999)