Ligation of CD44 by A3D8 Monoclonal Antibody Induces Terminal Differentiation of THP-1 Leukemia Cells by Promoting Cross-Talk between Extracellular Signal-Regulated Kinase and Phosphoinositide-3 Kinase/Akt Signaling Pathway.

Ligation of CD44 with anti-CD44 monoclonal antibody A3D8 induces terminal differentiation of human leukemia cells. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the importance of MEK/ERK, p38 MAPK, and phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in A3D8-induced monocytic differentiation of THP-1 leukemia cells. THP-1 cells displayed cytologic changes typical of mature monocytes and an increased expression of monocyte-specific antigen CD14 (49% ± 4%) and of myeloid-specific antigen CD11b (68% ± 6%) after 3 days of A3D8 treatment. The level of CD15 was also increased. The increase in the expression of these differentiation antigens was dose- and time-dependent. We found that CD44 ligation with A3D8 led to rapid and sustained activation of the essential kinases in the extracellular signal-regulated kinase pathway such as phospho-Raf-1, phospho-MEK1/2, and phospho-ERK1/2. However, the total levels of these kinases were not affected during the course of differentiation. Ser⁴⁷³ Akt phosphorylation was also observed shortly after the cells were exposed to A3D8 and sustained thereafter. In contrast, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and C-Jun N-terminal kinase were not increased by CD44 ligation. Pretreatment of cells with 20mM of MEK1 inhibitor PD98059 abrogated the A3D8-induced activation of MEK1/2 and ERK1/2 and potently inhibited the THP-1 differentiation. However, a p38 MAPK inhibitor SB203580 did not abolished the CD44 ligation-induced terminal differentiation of THP-1 cells. Pretreatment of cells with PI3-K inhibitor LY294002 resulted in a nearly complete inhibition of A3D8-induced terminal differentiation. Overexpression of dominant-negative Akt also reduced the A3D8-induced THP-1 differentiation, suggesting that A3D8-induced THP-1 differentiation is coupled to PI3-K/Akt activation. Surprisingly, the pharmacological inhibition of PI3-K blocked not only A3D8-induced Akt activation but also A3D8-induced activation of Raf, MEK and ERK pathway. By contrast, pretreatment of cells with PD98059 did not inhibit the A3D8-induced Akt activation, suggesting that PI3-K/Akt is the upstream of Raf/MEK/ERK pathway in A3D8-induced terminal differentiation of THP-1 leukemia cells. Taken together, our findings demonstrated that the cross-talk between the activation of A3D8-inducible PI3-K/Akt pathway and the Raf/MEK/ERK pathway in THP-1 cell exists, and plays a critical role during CD44 ligation-induced THP-1 differentiation.