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

Abstract

The role of insulin (INS), and insulin-like growth factor-I (IGF-I 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.

Numerous growth factors, cytokines, and chemokines are secreted by human CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner

“…The c-kit receptor positive (c-kit-R ϩ ) subset of CD34 ϩ cells was isolated by FACS as described. 19 Briefly, 2 ϫ 10 7 human A Ϫ T Ϫ MNCs were suspended in phosphate-buffered saline (PBS) supplemented with 5% bovine calf serum (BCS) and labeled for 30 minutes at 4°C with anti-kit-R monoclonal antibodies (clone No. 104D2) directly conjugated with Cy5 (Becton Dickinson, San Jose, CA) (20 L per 10 6 cells) and with an anti-CD34 monoclonal antibody directly conjugated with phycoerythrin (PE anti-HPCA-2) (Becton Dickinson) (20 L per 10 6 cells).…”

“…[19][20][21][22] A Ϫ T Ϫ MNCs were enriched for CD34 ϩ cells by an immuno-affinity selection with MiniMacs (Miltenyi Biotec, Auburn, CA) paramagnetic beads as previously described. 19,20 Mobilized peripheral blood (mPB) cells were obtained from granuloctye-colony stimulating factor (G-CSF)-mobilized breast cancer patients, and CD34 ϩ cells were isolated as described by us previously. 23 The purity of the isolated CD34 ϩ cells was greater than 97% as established by fluorescence-activated cell sorting (FACS) analysis ( Figure 1).…”

Biological significance of the expression of HIV-related chemokine coreceptors (CCR5 and CXCR4) and their ligands by human hematopoietic cell lines

“…30,31 Cells were infected at day 0 by BAL (R5 HIV) or HXB2 (X4 HIV). HIV strains (a gift of Dr F Gonzales-Scarano, University of Pennsylvania, Philadelphia, PA) were employed at a multiplicity of infection (MOI) of 0.02.…”

“…30,31 Cultures were performed in 96-well plates. The cells from the various hematopoietic cells lines (2 × 10 3 cells/ml) were resuspended in 1 ml of the culture medium without or in the presence of 5 ng (low dose) or 50 ng/ml (high dose) of the following chemokines: SDF-1␣, SDF-1␤, MIP-1␣, MIP-1␤, RANTES (R&D, Minneapolis, MN,USA).…”

Developmental Expression of Insulin Receptor Substrate-2 During Dimethylsulfoxide-Induced Differentiation of Human HL-60 Cells

“…In contrast, treatment of cells expressing IRS-2 with TNF-␣ does not affect IRS-2 tyrosine phosphorylation or tyrosine kinase activity of the insulin receptor. The emerging view is that the two IRS proteins have differences in function with IRS-1 likely to be more involved in cellular metabolism and growth while IRS-2 may be more important for leukocyte activation and function (12)(13)(14). Although IRS-2 is clearly involved in the activation pathways of a number of ILs, the factors that regulate expression of this protein are as yet unknown.…”