CaMKII regulates retinoic acid receptor transcriptional activity and the differentiation of myeloid leukemia cells

Abstract

Retinoic acid receptors (RARs) are members of the nuclear hormone receptor family and regulate the proliferation and differentiation of multiple different cell types, including promyelocytic leukemia cells. Here we describe a biochemical/functional interaction between the Ca 2+ /calmodulin-dependent protein kinases (CaMKs) and RARs that modulates the differentiation of myeloid leukemia cells. We observe that CaMKIIγ is the CaMK that is predominantly expressed in myeloid cells. CaMKII inhibits RAR transcriptional activity, and this enzyme directly interacts with RAR through a CaMKII LxxLL binding motif. CaMKIIγ phosphorylates RARα both in vitro and in vivo, and this phosphorylation inhibits RARα activity by enhancing its interaction with transcriptional corepressors. In myeloid cell lines, CaMKIIγ localizes to RAR target sites within myeloid gene promoters but dissociates from the promoter upon retinoic acid-induced myeloid cell differentiation. KN62, a pharmacological inhibitor of the CaMKs, enhances the terminal differentiation of myeloid leukemia cell lines, and this is associated with a reduction in activated (autophosphorylated) CaMKII in the terminally differentiating cells. These observations reveal a significant cross-talk between Ca 2+ and retinoic acid signaling pathways that regulates the differentiation of myeloid leukemia cells, and they suggest that CaMKIIγ may provide a new therapeutic target for the treatment of certain human myeloid leukemias. IntroductionHematopoiesis involves an intricate, functional interaction between lineage-specific cytokines and lineage-specific transcription factors. Among these transcription factors the retinoic acid receptors (RARs) are important regulators of myeloid lineage differentiation. These receptors are members of the ligand-activated nuclear receptor family and include 2 distinct families, the RARs and retinoid X receptors (RXRs), which bind as RAR/RXR heterodimers to their specific target sequences, the retinoic acid response elements (RAREs). RARs play a critical role in regulating myeloid differentiation, since retinoic acid (RA) stimulates the granulocytic differentiation of normal hematopoietic precursors (1), and knockout mice deficient in both RARα and RARγ exhibit a block to granulocyte differentiation (2). Moreover, a critical genetic event in the development of human acute promyelocytic leukemia (APL) involves the acquisition of a t(15;17) chromosome translocation resulting in the leukemogenic PML-RARα fusion protein that acts as a dominant-negative RAR to block normal myeloid differentiation (3,4). This differentiation block can be overcome with relatively high, pharmacological concentrations of RA, and the clinical use of RA to induce terminal differentiation of leukemia cells has had a remarkably beneficial impact on the therapy of APL (5, 6).Ligand binding to RAR/RXR alters its transcriptional activity by triggering a conformational change in this complex that inhibits its interaction with transcriptional corepressors while enhancing interaction ...

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

“…How this effect also caused a loss of chemotaxis is unclear, but these results support the notion that attenuated expression of CKLiK is important to normal neutrophil development, and suggest that CKLiK may antagonize the activation of neutrophil-specific transcription programs. Importantly, these results are consistent with the recently identified capacity of CaMK inhibitors to induce neutrophil differentiation and of CaMKIIγ to inhibit RAR-regulated differentiation of myeloid leukemic cell lines [15][16][17]. Identifying the precise mechanism by which CKLiK interferes with neutrophil gene expression will require more detailed analyses, but CKLiK may target a repressor of neutrophil differentiation, such as the CCAAT displacement protein (CDP/cut) or the transcription factor PU.1.…”

“…7B). EPRO cells exposed to KN-93 were also assessed for morphologic maturation similar to that found in MPRO cells exposed to KN-62 [16,17]. As shown in Figure 7C, a small but significant number of EPRO cells exposed to either KN-93 or KN-62 showed maturation based on changes in nuclear morphology.…”

“…HL-60 and NB4) towards mature neutrophils [15][16][17]. Furthermore, recent studies by Dr. Steven Collin's group demonstrated that CaMKIIγ directly inhibits RARα transcriptional activity [16,17]. Together these studies suggest that at least one CaMK, e.g.…”

“…The function of neutrophils is dependent on multiple calcium signaling pathways, and recent studies have demonstrated that CaMK inhibitors such as KN93, which interfere with Ca 2+ / CaM binding, can affect the proliferation and differentiation of human leukemic cells [15][16][17]. Since these inhibitors disrupt CaMKI and CaMKIV activities [32,33], and may therefore affect CKLiK activities in neutrophils, we tested whether KN-93 and the recently identified CaMKKα inhibitor STO-609 also affect the growth or functional responses of our cell lines.…”

“…However, other studies have demonstrated that CaMK inhibitors KN-93 and KN-62 can induce the differentiation of multiple leukemic myeloid cells (e.g. HL-60 and NB4) towards mature neutrophils [15][16][17]. Furthermore, recent studies by Dr. Steven Collin's group demonstrated that CaMKIIγ directly inhibits RARα transcriptional activity [16,17].…”