The role of insulin (INS) and insulin-like growth factor-I (IGF-I) in regulating human erythropoiesis. Studies in vitro under serum-free conditions--comparison to other cytokines and growth factors.
The role of insulin (INS), and insulin-like growth factor-I (IGF-I) in the regulation of human erythropoiesis is not completely understood. To address this issue we employed several complementary strategies including: serum free cloning of CD34+ cells, RT-PCR, FACS analysis, and mRNA perturbation with oligodeoxynucleotides (ODN). In a serum-free culture model, both INS and IGF-I enhanced survival of CD34+ cells, but neither of these growth factors stimulated their proliferation. The influence of INS and IGF-I on erythroid colony development was dependent on a combination of growth factors used for stimulating BFU-E growth. When BFU-E growth was optimally stimulated with erythropoietin (EpO) + kit ligand (KL) the large erythroid colonies developed normally even in the absence of INS or IGF-I. However, the addition of both of these growth factors slightly enhanced colony size. On the other hand, if erythroid colonies were stimulated suboptimally with EpO + IL-3 only, INS or IGF-I increased the number of small erythroid bursts by approximately 30%. Both INS and IGF-I activated signal transduction in maturing human erythropoietic cells as determined by phosphorylation of the insulin receptor substrate-2 (IRS-2) protein. We also found by RT-PCR that mRNA coding for INS-R is expressed in FACS sorted CD34+, c-kit-R+ marrow cells, and in cells isolated from BFU-E and CFU-GM colonies. Expression of INS-R protein on these cells was subsequently confirmed by cytofluorometry. In contrast, the receptor for insulin-like growth factor-I (IGF-IR) was not detected on CD34+ cells, and was first easily detectable on more differentiated cells derived from day 6 BFU-E and CFU-GM colonies. We conclude that INS and IGF-I may be survival factors for human CD34+ cells, but are not required during early erythropoiesis. In contrast, both growth factors may play some role at the final stages of erythroid maturation.
Ratajczak
J
J
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Zhang
Q
Q
Pertusini
E
E
Wojczyk
B S
BS
Wasik
M A
MA
Ratajczak
M Z
MZ
D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000494:pharmacology*; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004920:Erythropoiesis / Q000187:drug effects* / Q000502:physiology; D004921:Erythropoietin / Q000494:pharmacology; D005434:Flow Cytometry; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis*; D011972:Receptor, Insulin / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
D016895:Culture Media, Serum-Free; D016207:Cytokines; D006133:Growth Substances; D007328:Insulin; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D004921:Erythropoietin; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin