Growth factors produced by human embryonic kidney cells that influence megakaryopoiesis include erythropoietin, interleukin 6, and transforming growth factor-beta.
Partially purified protein preparations containing megakaryocyte growth factor activity were prepared from human embryonic kidney (HEK) cell conditioned medium using ammonium sulfate precipitation, Cibicron blue affinity chromatography, and wheatgerm lectin affinity chromatography. Treatment of these preparations with neutralizing antibodies directed against erythropoietin (EPO) and interleukin 6 (IL6) resulted in a dramatic reduction in their capacity to stimulate megakaryocyte maturation in vitro. The presence of EPO in these preparations was confirmed by both immunoblotting and use of a mouse spleen erythroid progenitor cell proliferation assay routinely used to quantitate EPO activity in vitro. Northern blot analysis of HEK cell-derived mRNA with IL6 DNA probes revealed the presence of an IL6 transcript with a molecular size of 1.3 kb. Analysis of the HEK cell-derived preparation by ELISA confirmed the presence of immunologically reactive IL6. In addition, it was shown that purified recombinant human EPO and IL6 stimulated megakaryocyte maturation in the in vitro assay used in this study. These data indicate that the activity in HEK cell conditioned medium that stimulates megakaryocyte maturation in vitro is predominantly due to the presence of IL6 and EPO. Immunoneutralization studies of another HEK cell-derived preparation, which was inhibitory in the megakaryocyte maturation assay, demonstrated that it contained transforming growth factor beta (TGF beta), a potent inhibitor of megakaryocyte maturation. Taken together, these studies indicate that HEK cell conditioned medium, which has previously been reported to contain megakaryocyte growth factor activity, is comprised of a complex mixture of growth and differentiation factors, some of which promote and others that inhibit the process of megakaryopoiesis.
Withy
R M
RM
Recombinant Protein Development and Molecular Biology Department, Genzyme Corporation, Framingham, Massachusetts 01701.
Rafield
L F
LF
Beck
A K
AK
Hoppe
H
H
Williams
N
N
McPherson
J M
JM
D001681:Biological Assay; D015152:Blotting, Northern; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000378:metabolism*; D006133:Growth Substances / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D008533:Megakaryocytes / Q000166:cytology*; D012333:RNA, Messenger / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D006133:Growth Substances; D015850:Interleukin-6; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D004921:Erythropoietin