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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 1993-1999
Identification of a family of alternatively spliced mRNA species
of angiopoietin-1
Yao-Qi Huang,
Jian-Jun Li, and
Simon Karpatkin
From the New York University School of Medicine and Kaplan Cancer
Center, New York, NY.
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.
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