p38 and ERK MAPKs Differentially Regulate Expansion and Differentiation of Human Cord Blood Derived CD34⁺ Cells during Neutropoiesis.

Aberrant regulation of myelopoiesis can lead to severe clinical conditions ranging from myeloproliferative disorders to severe immune deficiencies. It is therefore important to understand the molecular mechanisms underlying these processes. Mitogen-activated protein (MAP) kinase cascades regulate a variety of cellular functions, including differentiation, proliferation, and apoptosis. Three major groups of MAP kinases exist including the p38 MAP kinase family, the ERK family and JNK kinase family. ERK is activated through phosphorylation by MEK in response to several cytokines and growth factors, and primarily mediates mitogenic and anti-apoptotic signals. Members of the p38 family of MAP kinases are activated by MKK3/MKK6, primarily by stress stimuli, but also by various cytokines and have shown to be involved in regulation of apoptosis, cell-cycle arrest and cell differentiation. Although both the ERK and p38 pathways have been demonstrated to regulate proliferation and differentiation in a variety of cell types, their role in regulating hematopoietic cell fate decisions remains unclear.

In this study we have utilized an ex-vivo human CD34⁺ culture system to investigate neutropoiesis. During G-CSF-induced differentiation, cells were incubated either in absence or presence of the specific p38 inhibitor SB203580 or MEK inhibitor U0126. Proliferation was measured by counting cells every 3 days and percentages of apoptotic cells were determined by analyzing Annexin-V positive cells. Inhibition of p38 during neutrophil differentiation by SB203580 resulted in increased progenitor expansion, which was not due to enhanced survival. Inhibition of p38 also resulted in higher percentages of neutrophils with banded or segmented nuclei as analyzed by May-Grunwald Giemsa staining, however we observed no effects on the formation of CFU-GM colonies. In contrast to p38, inhibition of ERK activity resulted in a dramatic decrease in expansion, which again was not the result of an increase in apoptosis. Interestingly, after 17 days of neutrophil differentiation, similar numbers of mature neutrophils in both U0126 treated and control cells were found. Moreover, treatment of cells with U0126 resulted in increased CFU-GM colony formation suggesting a delay in early differentiation. Retroviral transduction experiments were performed to ectopically express constitutively-active MKK3. Ectopic MKK3 expression resulted in a clear reduction in progenitor number, which was not due to increased apoptosis. Furthermore, transduction of cells with constitutively-active MKK3 resulted in a dramatic block in neutrophil differentiation. Interestingly, whereas neutrophil development was inhibited, the percentage of monocytic cells was induced in cells expressing constitutively-active MKK3.

These results demonstrate the importance of both ERK and p38 MAP kinases in regulating neutrophil development. Inhibition of p38 activity is necessary for terminal differentiation of CD34⁺ progenitor cells, regulating expansion as well as differentiation, whereas ERK activity is primarily required for optimal expansion of progenitors during neutropoiesis. Modulation of these kinases may provide novel therapeutic strategies for a variety of clinical disorders where normal stem cell function is perturbed.