Norimichi Ochi

Molecular Cloning and Expression of Megakaryocyte Potentiating Factor cDNA

The human megakaryocyte potentiating factor (hMPF) has been previously purified from a culture supernatant of human pancreatic cancer cells HPC-Y5 (Yamaguchi, N., Hattori, K., Oh-eda, M., Kojima, T., Imai, N., and Ochi, N.(1994) J. Biol. Chem. 269, 805-808). We have now isolated hMPF cDNA from a HPC-Y5 cDNA library using polymerase chain reaction and plaque hybridization methods. The hMPF cDNA encodes a polypeptide consisting of 622 amino acids, including a signal peptide of 33 amino acids, and with a deduced molecular mass of 68 kDa, although HPC-Y5 cells secrete a 33-kDa form of hMPF. Human MPF does not show any significant homology with other previously described sequences. The cDNA was expressed in COS-7 and Chinese hamster ovary (CHO) cells, and megakaryocyte potentiating activity was detected in their culture supernatant. The COS-7 cells secreted only a 33-kDa recombinant hMPF, whereas an additional 30-kDa form was detected in the culture medium of CHO cells. The 33-kDa rhMPF purified from CHO cells showed megakaryocyte potentiating activity, but not the purified 30-kDa rhMPF. The difference in structure and activity between the 33- and 30-kDa forms of hMPF was ascribed to the existence in the 33-kDa form of the C-terminal 25 amino acid residues.

Thrombopoietic activity of human interleukin-6

Thrombopoietin (TPO), a regulatory factor in platelet production, was purified from the conditioned medium of TNK‐01 cells cultured in the presence of human interleukin‐1. The N‐terminal sequence of purified TPO was determined to be VPPGEDSKDVAAPHRQPLT, identical to that of the N‐terminal region of human interleukin‐6 (IL‐6). Two forms of TPO with molecular masses of 24 and 27 kDa were identified as IL‐6 by Western analysis using an anti‐IL‐6 antibody. Commercial recombinant human IL‐6 produced in Escherichia coli, stimulated megakaryocyte colony formation in the presence of mouse interleukin‐3 and increased the number of peripheral platelets in mice in a dose‐dependent manner. From these results, it is concluded that human IL‐6 has thrombopoietic activity.