Regulation of the hematopoietic cell kinase (HCK) by PML/RARα and PU.1 in acute promyelocytic leukemia

doi:10.1016/j.leukres.2011.09.012

Leukemia Research

Volume 36, Issue 2, February 2012, Pages 219-223

https://doi.org/10.1016/j.leukres.2011.09.012 Get rights and content

Abstract

This study investigates the dynamic regulation of human hematopoietic cell kinase (HCK) in acute promyelocytic leukemia (APL) and the underlying molecular mechanisms. First, the level of HCK in APL blasts was found lower than that in normal granulocytes and monocytes. Second, the HCK promoter was repressed by PML/RARα and this repression required PU.1. PU.1 was capable of transactivating the HCK promoter through a region encompassing three PU.1 motifs. Chromatin immunoprecipitation assays provided evidence that PU.1 and PML/RARα bound to the HCK promoter in vivo. Finally, we found an unequivocal increase of HCK expression upon treatment with all-trans retinoic acid.

Introduction

Acute promyelocytic leukemia (APL) is characterized by a translocation between chromosomes 15 and 17, producing the promyelocytic leukemia-retinoic acid receptor α (PML/RARα) fusion protein, which plays an essential role in the leukemogenesis of APL. Immature promyelocytic blasts are accumulated in patient's bone marrow and peripheral blood. Pharmacological concentrations of all-trans retinoic acid (ATRA) can promote differentiation of leukemic cells and induce APL blasts to terminal differentiation. ATRA causes the dissociation of corepressor complexes from PML/RARα, leading to re-activation of genes repressed by PML/RARα [1]. The PML/RARα fusion protein contains the protein–protein interaction domain of PML and the DNA binding domain of RARα, enabling the direct and indirect regulations of downstream targets. Our recent study has determined a critical mechanism of differentiation blockade in APL, by which PML/RARα disrupts the function of the hematopoietic transcription factor PU.1 and subsequently blocks the downstream PU.1 signaling [2].

Hematopoietic cell kinase (HCK), one member of the Src family of tyrosine kinases (SFKs), is restricted to hematopoietic cells. HCK expression is gradually increased with the myeloid differentiation process and reaches the highest level in fully differentiated cells of granulocytic and monocytic lineages [3], suggesting its possible role in hematopoietic differentiation. Generally, constitutively activated SFKs contribute to cell transformation. For example, in chronic myeloid leukemia (CML), a type of Philadelphia chromosome-positive (Ph+) leukemia, HCK interacts with and is activated by the BCR/ABL fusion protein. Activated HCK subsequently phosphorylates STAT5 and triggers activation of the signaling pathway downstream of STAT5, which plays a critical role in BCR/ABL-mediated transformation of myeloid cells [4]. However, emerging evidence shows HCK could act as a potential tumor suppressor in several types of leukemia. For instance, in acute myeloid leukemia (AML) with inv(16)(p13q22) (M4Eo, FAB), the HCK gene has a significantly lower expression level compared to other AML-M4 cases. HCK inhibition also occurs in Ph(−) acute lymphocytic leukemia (ALL) due to aberrant methylation of the CpG island in the HCK promoter [5]. These observations indicate that HCK may have potential tumor suppressor properties. This concept is also reinforced by other reports, wherein HCK has been shown to trigger apoptosis. HCK can interact with guanine nucleotide exchange factor C3G and functionally induce an apoptotic pathway dependent on the catalytic activity of HCK [6]. The SH3 domain of HCK also mediates signaling at the plasma membrane, thus triggering a pathway leading to caspase-3 dependent cytochrome c release and apoptosis [7]. All these findings suggest that impaired HCK expression may be involved in the pathogenesis of leukemia.

In this study, we examined the transcriptional regulation of HCK in the pathogenesis and treatment of APL. We found a lower level of HCK in APL patients, which was regulated by PML/RARα. Furthermore, we showed that the repression required the presence of PU.1 and that the three PU.1 motifs embedded in the HCK promoter contributed to PU.1-mediated transactivation and subsequent repression by PML/RARα. Finally, our results revealed that ATRA enhanced HCK expression in APL cells. Taken together, this study highlights the regulatory mechanism of HCK and the importance of HCK as a tumor suppressor in APL.

Section snippets

Cell culture

U937, PR9 and NB4 cells were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS) (GIBCO). 293T cells were maintained in DMEM medium (GIBCO) supplemented with 10% FBS. Cells were grown in an incubator at 37 °C and 5% CO₂. ZnSO₄, ATRA and cycloheximide (Sigma) were used at final concentrations of 100 μM, 1 μM and 10 mg/ml, respectively.

Plasmid constructions and site-directed mutagenesis

An approximately 1.1 kb DNA segment upstream of the HCK transcription start site was cloned into pGL4-basic vector (Promega). Mutations

HCK is repressed by PML/RARα in APL cells, and the repression is dependent on PU.1 expression in cells

HCK is predominantly expressed in monocytic and granulocytic hematopoietic cells and is up-regulated during myeloid differentiation, which indicates that HCK plays a substantial role in the differentiation process of hematopoietic cells. We were interested in the HCK expression level in APL patients. We first retrieved the expression profiles of 17 APL patients and 12 normal bone marrow specimens containing 7 cases of monocytes and 5 cases of granulocytes [9], [10], [11], [12], [13], [14] and

Discussion

In this study, we elucidated an important role for the transcription factor, PU.1, in HCK transcriptional regulation. Since PU.1 is a critical transcriptional regulator of hematopoiesis, many PU.1 targets play key roles in proliferation, survival and differentiation processes of monocytic and granulocytic lineages [19]. Macrophages and granulocytes do not develop in PU.1-null mutant mice, confirming the crucial role of PU.1 in terminal myeloid differentiation [20]. Remarkably, HCK expression is

Conflict of interest statement

None of the authors declare a conflict of interest.

Acknowledgments

The authors would like to thank Dr. Pelicci for the gift of the PR9 cell line. This work was supported in part by National Natural Science Foundation Grants (90919059 and 30730033) and Ministry of Science and Technology of China Grants (2009CB825607 and 2011CB910202).

Contributions. DZ, XY: acquisition, analysis and interpretation of data, drafting of the article; YT, PW, XZ, WY and XJ: analysis and interpretation of data; revising the article; JZ and KW: conception of the study, analysis and

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Regulation of the hematopoietic cell kinase (HCK) by PML/RARα and PU.1 in acute promyelocytic leukemia

Abstract

Introduction

Section snippets

Cell culture

Plasmid constructions and site-directed mutagenesis

HCK is repressed by PML/RARα in APL cells, and the repression is dependent on PU.1 expression in cells

Discussion

Conflict of interest statement

Acknowledgments

Blood

Cancer Cell

J Biol Chem

J Biol Chem

Cell

Blood

Immunity

Cell

Immunity

Blood

The Src family kinase Hck couples BCR/ABL to STAT5 activation in myeloid leukemia cells

EMBO J

Aberrant DNA methylation of the Src kinase Hck, but not of Lyn, in Philadelphia chromosome negative acute lymphocytic leukemia

Leukemia