Protein Kinase C α Controls Erythropoietin Receptor Signaling

Abstract

Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent "renewal divisions" induced in the presence of Epo, stem cell factor, and dexamethasone.

Apoptotic mechanisms in the control of erythropoiesis

“…130 The activation of PKB/AKT following Epo stimulation requires a normal protein kinase C (PKC) activity, as suggested by the experiments carried out with PKC inhibitors. 131 As mentioned above, the FOXO family of Forkhead transcription factors is an important target of the activated PKB/AKT: activated PKB/AKT directly phosphorylates FKHR-L1 and other forkhead transcription factors resulting in cytoplasmic retention and inactivation, thus inhibiting the activity of these transcription factors required for the control of the expression of several genes involved in cell cycle control, cell death and oxidative stress. 132 There is evidence that the activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal.…”

The majority of the in vitro erythroid expansion potential resides in CD34- cells, outweighing the contribution of CD34+ cells and significantly increasing the erythroblast yield from peripheral blood samples

“…We describe a culture method modified from Leberbauer et al (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34 + cells. This pure population of immature erythroblasts can be expanded to obtain 4¥10 8 erythroblasts from 1¥10 8 PBMC after 13-14 days in culture. Upon synchronized differentiation, high levels of enucleation (80-90%) and low levels of cell death (<10%) are achieved.…”

“…-hematopoietic stem cells are present in cord blood, peripheral blood and bone marrow 8,9 and support hematopoiesis/erythropoiesis. 10 We hypothesized that these CD34 -cells may make a substantial contribution to erythroid expansion when total PBMC are used as starting material for the in vitro culture of erythroblasts.…”

Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (-) cells (mutations Chartres 1 and 2)

“…26,31 Immunoprecipitations and total cell lysates Cells were lysed as described 32 and quantified by Lowry assay (Biorad, Hercules, CA, USA). For immunoprecipitations 1.5×10 7 erythroblasts or 5×10 7 erythrocytes were pre-cleared (1h) with unbound protein-G-beads.…”