Elevated Cyclin E Levels, Inactive Retinoblastoma Protein, and Suppression of the p27^KIP1 Inhibitor Characterize Early Development of Promyeloid Cells into Macrophages

Abstract

Cyclin-dependent kinase inhibitors such as p27^KIP1 have recently been shown to lead to cellular differentiation by causing cell cycle arrest, but it is unknown whether similar events occur in differentiating promyeloid cells. Hematopoietic progenitor cells undergo lineage-restricted differentiation, which is accompanied by expression of distinct maturation markers. Here we show that the classical growth factor insulin-like growth factor I (IGF-I) potently promotes vitamin D₃-induced macrophage differentiation of promyeloid cells, as assessed by measurement of a coordinate increase in expression of the integrin α subunit CD11b, the CD14 lipopolysaccharide receptor, and the macrophage-specific esterase, α-naphthyl acetate esterase, as early as 24 h following initiation of terminal differentiation. Addition of IGF-I to cells undergoing vitamin D₃-induced differentiation also leads to an early increase in expression of cyclin E, phosphorylation of the retinoblastoma tumor suppressor protein, and a doubling of the cell number. Early expression of CD11b (24 h) is simultaneously accompanied by inhibition in the expression of p27^KIP1. Cell cycle analysis with propidium iodide revealed that CD11b expression at 24 h following initiation of differentiation occurs at all phases of the cell cycle instead of only those cells arrested in G₀/G₁. Similarly, development of a novel double-labeling intra- and extracellular flow-cytometric technique demonstrated that single cells expressing the mature leukocyte differentiation antigen CD11b can also incorporate the thymidine analog bromodeoxyuridine. Likewise, expression of the intracellular DNA polymerase δ cofactor/proliferating-cell nuclear antigen at 24 h is also simultaneously expressed with the surface marker CD11b, indicating that these cells continue to proliferate early in their differentiation program. Finally, at 24 h following induction of differentiation, IGF-I promoted a fourfold increase in the uptake of [³H]thymidine by purified populations of CD11b-expressing cells. Taken together, these data demonstrate that the initial steps associated with terminal macrophage differentiation occur concomitantly with progression through the cell cycle and that these very early differentiation events do not require the accumulation of p27^KIP1.

ACKNOWLEDGMENTS

We gratefully acknowledge the instrumentation and expert technical assistance provided by Gary Durack in the University of Illinois Urbana-Champaign Biotechnology Center Flow Cytometry Facility.

This research was supported by grants to K.W.K from the National Institutes of Health (AG-06246, DK-49311, and MH-51569) and the Pioneering Research Project in Biotechnology financed by the Japanese Ministry of Agriculture, Forestry and Fisheries and to G.G.F. from the National Institutes of Health (CA 61931). The UIUC Biotechnology Center Flow Cytometry Facility was supported by NIH grant PHS 1S10 RR02277.