Induction of GD3/α1-adrenergic receptor/transglutaminase 2-mediated erythroid differentiation in chronic myelogenous leukemic K562 cells

Abstract

The disialic acid-containing glycosphingolipid GD3 recruited membrane transglutaminase 2 (TG2) as a signaling molecule for erythroid differentiation in human chronic myelogenous leukemia (CML) K562 cells. The α1-adrenergic receptor (α1-AR)/TG2-mediated signaling pathway regulated GD3 functions, including gene expression and production, to differentiate CML K562 cells into erythroid lineage cells. Epinephrine, an AR agonist, increased membrane recruitment as well as GTP-photoaffinity of TG2, inducing GD3 synthase gene expression. Epinephrine activated PI3K/Akt signaling and GTPase downstream of TG2 activated Akt. The coupling of TG2 and GD3 production was specifically suppressed by prazosin (α1-AR antagonist), but not by propranolol (β-AR antagonist) or rauwolscine (α2-AR antagonist), indicating α1-AR specificity. Small interfering RNA (siRNA) experiment results indicated that the α1-AR/TG2-mediated signaling pathway activated PKCs α and δ to induce GD3 synthase gene expression. Transcription factors CREB, AP-1, and NF-κB regulated GD3 synthase gene expression during α1-AR-induced differentiation in CML K562 cells. In addition, GD3 synthase gene expression was upregulated in TG2-transfected cells via α1-AR with expression of erythroid lineage markers and benzidine-positive staining. α1-AR/TG2 signaling pathway-directed GD3 production is a crucial step in erythroid differentiation of K562 cells and GD3 interacts with α1-AR/TG2, inducing GD3/α1-AR/TG2-mediated erythroid differentiation. These results suggest that GD3, which acts as a membrane mediator of erythroid differentiation in CML cells, provides a therapeutic avenue for leukemia treatment.

Keywords: adrenergic receptor; erythroid differentiation; ganglioside GD3; human chronic myelogenous leukemia K562 cell; transglutaminase 2.

Figure 1. Schematic illustration of disialyl ganglioside GD3 synthesis in mammalian cells

Figure 2. Increase in membrane recruitment of TG2 in response to epinephrine

First, 50 μg of membrane proteins were isolated and subjected to 10% SDS-PAGE, as described in Experimental Procedures. TG2 expression levels were determined by Western blot analysis. RT-PCR analysis of GD3 synthase mRNA. Total RNAs were isolated from K562 cells after 0, 0.5, 1, 2, 3, or 4 days of treatment with 10 μM epinephrine. Then 1 μg of total RNA from each cell was subjected to RT-PCR. β-Actin indicates that equal amounts of RNA were loaded in each lane. Data are representative of three experiments (means ± SD). *P < 0.05 vs. control (0).

Figure 3. Activation of Akt phosphorylation and inactivation of ERK1/2 by treatment with 10 μM epinephrine and comparison of the GTP photoaffinity of membrane-bound TG2 and its transglutaminase activity at varying calcium concentrations

(A) Activation of Akt phosphorylation and inactivation of ERK1/2 following treatment with 10 μM epinephrine. Cells were incubated with 10 μM epinephrine for the time periods indicated in the figure. Then, 25 μg of protein was subjected to 10% SDS-PAGE. Phospho-specific antibodies were used to measure the activation of Akt and ERK1/2. The blots were stripped and then reprobed with anti-Akt and anti-ERK1/2 antibodies. Data are representative of three experiments. (B) Influence of GTP-bound TG2 on Akt phosphorylation following incubation with 10 μM epinephrine for 2 days at different Ca2+ concentrations. The GTP binding activity of membrane-bound TG2 was determined using affinity labeling with radioactive GTP, as described in Experimental Procedures. [α-³²P]GTP-bound TG2 was immunoprecipitated with anti-TG2 antibody. The bound radiolabeled GTP was visualized by autoradiography, following 10% SDS-PAGE. GAPDH indicated that an equal amount of proteins was loaded in each lane (lower panel). Data are representative of three experiments. (C) TGase activities in the cytosolic fraction were measured by incubation with 10 μM epinephrine for 2 days at different Ca²⁺ concentrations. Data are representative of three experiments (means ± SD). *P < 0.05 and **P < 0.01, vs. control (0).

Figure 4. α1-AR-mediated increases in GD3 expression and membrane recruitment of TG2

(A) Ganglioside profiles. Gangliosides were isolated from 1 × 10⁹ K562 cells. Separated gangliosides were revealed by spraying with the resorcinol hydrochloride reagent (upper panel). Immunostaining of GD3 with a GD3-specific antibody (Lower panel). (B) α1-AR-specific GD3 synthase expression as measured by RT-PCR. The cells were incubated for 2 days with 10 μM epinephrine in the presence of the combination containing the AR antagonists prazosin (α1), rauwolscine (α2), and propranolol (β). (C) Levels of membrane-bound TG2 in response to epinephrine in the presence of several AR antagonists. Total TG2 level indicates that equal amounts of protein were loaded. M (marker) denotes a series of gangliosides (GM3, GM2, GM1, GD3, and GD1a). Data are representative of three experiments (means ± SD). *P < 0.05 vs. control (0).

Figure 5. PKC α and δ mediated GD3 synthase expression via α1-AR/TG2

K562 cells were incubated with 10 μM epinephrine in the presence of the combination containing rauwolscine and propranolol for 2 days. (A) Membrane translocation of PKC α and PKC δ by α1-AR. (B) Down-regulation of GD3 synthase expression and membrane recruitment of TG2 following treatment with the PKC α and PKC δ inhibitors, Gö6976 and Rottlerin, respectively. (C) Inhibitory effect of TG2 and GD3 synthase siRNAs on the activation of PKC α and PKC δ by α1-AR. Data are representative of three experiments (means ± SD). *P < 0.05 vs. control (0).

Figure 6. Promoter assay of GD3 synthase and EMSA analysis

Cells were incubated with 10 μM epinephrine and rauwolscine or propranolol. (A) The promoter region of GD3 synthase. The region from −2690 to −690 contains two AP-1 (consensus sequence motif: 5′-TGACG-3′), four CREB (5′-TGACGTCA-3′), one SP-1 (5′-GGGTGG-3′), and one NF-κB (5′-GGGAGACCT-3′) putative regulatory elements. (B) Analysis of deletion mutants of the GD3 synthase gene promoter in K562 cells in response to 10 μM epinephrine. pGL3-basic (without any promoter or enhancer) was used as a negative control. pGL3-control (with the SV40 promoter and enhancer) was used as a positive control. Each construct was co-transfected into K562 cells with pCMV as the internal control. The transfected cells were incubated in the presence (solid bars) or absence (open bars) of 10 μM epinephrine for 2 days. Relative luciferase activity was normalized to β-galactosidase activity derived from pCMV. (C) Electrophoretic mobility shift assay (EMSA). Nuclear protein fractions were isolated after incubation with or without 10 μM ephinephrine for 2 days. DNA–protein complexes were analyzed on a 4% non-denaturing polyacrylamide gel. For competition experiments, a 50-fold molar excess of unlabeled wild-type oligonucleotides (C) or unlabeled mutant oligonucleotides (M) was used. The results were obtained from three independent experiments.

Figure 7. GD3 synthase expression and expression of several erythroid differentiation marker genes induced by α1-AR/TG2-mediated signaling

K562 cells were transfected with GD3 synthase (GD3S) and TG2 cDNAs. Without α1-AR antagonist prazosin, cells were treated with α2-AR and β-AR-specific antagonists such as rauwolscine and propranolol in the presence of 10 μM epinephrine, classified as +Epi+Raw+Prop. Then, 1 μg of total RNA isolated from K562 cells was subjected to RT-PCR using primers specifically designed for megakaryotic or several erythroid lineage marker genes, as described in Experimental Procedures. β-Actin mRNA expression indicated that equal amounts of mRNA were used for RT-PCR in each lane. (A) RT-PCR of GD3 expression induced by α1-AR/TG2-mediated signaling. (B) Induction of several differentiation marker genes by α1-AR/TG2-mediated signaling. (C) Cells were incubated with 10 μM epinephrine for 2 days and benzidine staining was performed as described in the ‘Materials and Methods’ section. Data are representative of three experiments (means ± SD). *P < 0.05 vs. control (0).

Figure 8. Schematic diagram of GD3 synthase expression induced by α1-AR/TG2-mediated signaling in K562 cells

PLC, phospholipase C; PKC, protein kinase C; DAG, diacylglycerol.