RCAS1; A novel regulator of apoptosis pF erythroid progenitor cells

Erythropoiesis is closely regulated by an interacting network of growth factors and cytokines, which affect apoptosis of erythroid progenitor cells. Although Fas ligand and TRAIL have been identified as inducers of apoptosis in erythroid progenitor cells, he mechanisms by which apoptosis is controlled during erythropoiesis may still be incompletely recognized. We investigated the role of a novel tumor-associated antigpn, RCAS1 (receptor binding cancer antigen expressed on SiSo cells) in the regulation of apoptosis of erylhroid progenitor cells, using erythroid colony forming cells (ECFC) purified from human peripheral blood. RCAS1 is a type II membrane protein isola :ed from the human uterine adenocarcinoma cell line, SiSo, and is reported to induce apoptcsis of activated T lymphocytes(l). Binding experiments involving RCAS1 using recombinmt RCAS1-GST fusion protein showed that the majority of ECFCs expressed abundmt receptors for RCAS1 (RCAS1R), and further that the expression diminished rapMly during erythroid maturation in vitro (day 7 ECFCs 96.6% vs. dayl3 ECFCs 4.8%). When the soluble form of RCAS1 was added to the cultures, the percentage of apoptotic cells, determined by annexin V binding in 10 experiments (annexin VVPItand) was 37.9±9.4% after incubation for 24 hours with nRCASl, compared to 10.0+1.7% in control cultuires (p<0.001). An active intracellular apoptotic process including a collapse of mitochondria! transmembrane potential, as well as activation of caspaseS and 3 was observed in cells cultured with nRCASl. Neither the addition of anti-Fas blocking antibody (clone; 4B43B) nor that of Fas-Fc reduced RCAS 1-induced apoptosis. Analysis of the binding! of RCAS1 to bone marrow cells from a normal volunteer reveraled the presence of RCASJ1R on the cells with an immature erythroid phenotype (transferrin receptor /glycophorin| A). Furthermore, histochemical staining demonstrated RCAS1 expression in the cytoplasm of bone marrow macrophages. These findings indicate that RCAS 1, which is primarily produced by macrophages in hematopoietic tissue, may have a crucial role in controlling erythropoiesis, by modulating apoptosis of erythroid progenitor cells via a Fsindependent mechanism. (1) M. Nakashima, et al. Nat. Med. 1999; 5: 938-942.