For Peer Review

PLCβ1a and PLCβ1b selective regulation and cyclin D3 modulation reduced by Kinamycin F during K562 cell

differentiation

Journal: Journal of Cellular Physiology

Manuscript ID: JCP-14-0440

Wiley - Manuscript type: Original Research Article

Date Submitted by the Author: 15-Jul-2014

Complete List of Authors: Bavelloni, Alberto; Rizzoli Institute, Cell Biology Dmitrienko, Gary; University of Waterloo, Department of Chemistry,School of Pharmacy Goodfellow, Valerie; University of Waterloo, Department of Chemistry Ghavami, Ahmad; University of Waterloo, Department of Chemistry Piazzi, Manuela; University of Bologna, Departmentof Biomedical Science Blalock, William; Rizzoli Orthopedic Institute, Cell Biology

Chiarini, Francesca; CNR-National Research Council of Italy, Institute of Molecular Genetics Cocco, Lucio; University of Bologna, Human Anatomical Sciences Faenza, Irene; University of Bologna, Departmentof Biomedical Science

Key Words: Phospholipase C, cyclin D3, kinamycin F, nucleus, erythropoiesis

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PLCβ1a and PLCβ1b selective regulation and cyclin D3 modulation reduced by Kinamycin F

during K562 cell differentiation

Alberto Bavelloni 1,2

, Gary I. Dmitrienko5,6

, Valerie J. Goodfellow,5 Ahmad Ghavami

5, Manuela

Piazzi3 , William Blalock

4 , Francesca Chiarini

4 , Lucio Cocco

3,^, Irene Faenza

3,§

1SC Laboratory of Musculoskeletal Cell Biology, Rizzoli Orthopedic Institute, Bologna, Italy;

2Laboratory RAMSES, Rizzoli Orthopedic Institute, Bologna, Italy

3Cell Signaling Laboratory, Department of Biomedical Sciences, University of Bologna, Bologna,

Italy;

4CNR-National Research Council of Italy, Institute of Molecular Genetics, Bologna, Italy;

5Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

6School of Pharmacy, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

§ Correspondence to: Irene Faenza, email: irene.faenza2@unibo.it and

Lucio Cocco, email: lucio.cocco@unibo.it

^L.C. contributed vital new reagents, analyzed and interpreted the data, and helped draft the output.

Key words: PLCβ1, cyclin D3, Kinamycin F, γ-globin, erythropoiesis, nucleus

Running title: PLCβ1 regulation by kinamycin F

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Abstract

Here we report that both PLCβ1a and PLCβ1b are relevant regulators of erythropoiesis in that

kinamycin F, a potent inducer of γ-globin production in K562 cells, caused a selectively reduction

of both PLCβ1 isozymes even though the results point out that the effect of the drug is mainly

directed towards the expression of the PLCβ1a isoform. We have identified a different role for the

two isozymes as regulators of K562 differentiation process induced by kinamycin F. The

overexpression of PLCβ1b induced an increase in γ-globin expression even in the absence of

kinamycin F. Moreover during K562 differentiation, cyclin D3 level is regulated by PLCβ1

signaling pathway. Namely the amplification of the expression of the PLCβ1a, but not of PLCβ1b,

is able to maintain high levels of expression of cyclin D3 even after treatment with kinamycin F.

This could be due to their different distribution in the cell compartments since the amount of

PLCβ1b is mainly present in the nucleus in respect to PLCβ1a. Our data indicate that the

amplification of PLCβ1a expression, following treatment with kinamycin F, confers a real

advantage to K562 cells viability and protects cells themselves from apoptosis.

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Introduction

PLC isozymes receive and coordinate multiple upstream inputs, many of which involve Ras

superfamily GTPases (Harden et al., 2009). Phospholipase Cβ1 signaling and its activity is involved

in proliferative, mitogenic and differentiative events in the cell. PLCβ1 catalyzes the hydrolysis of

the signaling lipid phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 to produce the second

messengers inositol 1,5,-trisphosphate and diacylglycerol and is localized in both at the plasma

membrane and in the nucleus (Faenza et al., 2013). The nucleus has a phosphoinositol lipid

signaling pathway that is independent of the one found on the plasma membrane and PLCβ1 is one

of the major PLCs in the nucleus. There are two PLCβ1 splice variants (Bahk et al., 1998; Faenza et

al., 2000; Yang et al., 2013), PLCβ1a and PLCβ1b which have been detected also in the nucleus,

where they contribute to the regulation of cell cycle progression, in particular the G1/S transition

(Bahk et al., 1998) (Fiume et al., 2012) (Faenza et al., 2008) (O'Carroll et al., 2009). PLCβ1a and

PLCβ1b differ at the extreme C terminus, beyond the last residue observed in the reported crystal

structures. PLCβ1a is longer and contains a consensus postsynaptic density protein/Drosophila disc

large tumor suppressor/zona occludens-1protein motif at its C terminus, whereas PLCβ1b contains a

proline-rich region (Bahk et al., 1994). It is worthwhile mentioning that PLCβ1b is the one almost

entirely nuclear (Yang et al., 2013) (Bahk et al., 1998). K562 cells are a human erythroleukemic cell

line capable of differentiation into erythroid cells and expression of hemoglobin. Differentiated

K562 cells synthesize embryonic and fetal but not adult hemoglobin although they bear an intact h-

globin gene (Fordis et al., 1984). To some respect, K562 cells behave like thalassemic cells and

serve as a suitable model for hemoglobin ‘‘switching” (Tsiftsoglou et al., 2003). Differentiation of

K-562 cells is associated with an increase of expression of embryo–fetal globin genes, such as the ζ-

, ϵ-, and γ-globin genes. Using this system, several anti-cancer agents have also been characterized

with respect to activation of erythroid functions, such as mithramycin, cisplatin, and tallimustine

(Bianchi et al., 2001; Bianchi et al., 2000; Bianchi et al., 1999; Chiarabelli et al., 2003). We used

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human erythroleukemia K562 cells as an in vitro model of erythropoiesis because they have been

proven to be a powerful tool for investigating erythroid cell development. Kinamycin F has been

recently described as a potent differentiating agent of human erythroleukemia cells. In fact, it has

been demonstrated that treatment of K562 cells with kinamycin F induced erythroid differentiation,

a rapid apoptotic response, induction of caspase-3/7 activities and a delayed cell cycle progression

through the S and G2/M phases (O'Hara et al., 2010) . The bacterial metabolite kinamycin F, which

is being investigated as a potent antitumor agent, contains an unusual and potentially reactive diazo

group as well as a paraquinone and a phenol functional group (O'Hara et al., 2007) (Chen et al.,

2008). A potential target of kinamycin F is Cyclin D3 in that kinamycin F caused a selective

reduction of cyclin D3 protein at the level of transcription. Indeed previous studies have identified

cyclin D3 as one of the targets of PLCβ1 signal transduction (Cocco et al., 2006). More detailed

investigations showed that nuclear PLCβ1 activates cyclin D3 promoter during the differentiation

process. Since it has been recently shown that the antibacterial drug kinamycin F is able to induce

erythroid differentiation in K562 cells through the control of transcript levels of cyclin D3, we

wondered whether this regulation could be mediated by PLCβ1. Thus the aim of this study was to

determine the functional role of PLCβ1 in erythroleukemia K562 cells, trying to identify its targets

and its nuclear regulators after the anticancer drug kinamycin F treatment. We investigated the

effect of the antibiotic kinamycin F on the expression of PLCβ1 and its downstream effectors of the

signal transduction pathway through modulation of the expression of both PLCβ1a and -β1b.

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Materials and methods

Cell culture and differentiation induction

Human erythroleukemia cells (K562) were grown in RPMI 1640 (Sigma Aldrich) supplemented

with 10% heat-inactivated fetal bovine serum (FBS) and L-glutamine/streptomycin (1x) at an

optimal cell density between 3-5x105 . Cells (5x10

7) were differentiated with kinamycin F (2, 5 or 5

µM) and harvested after the indicated times of differentiation (16 or 24 h).

Nuclei isolation, total protein extraction, and Western blot analysis

A hypotonic shock combined with nonionic detergent (10mM Tris-Cl, pH 7.8; 1% Nonidet P-40),

essentially as described by Faenza et al. ((Faenza JBC 2000 )), has been used. Briefly, 5 x 106 cells

were lysed in 400 µl nuclear isolation buffer [10 mM Tris-HCl (pH 7.8),1% Nonidet P-40, 10 mM

β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg aprotinin and leupeptin/ml, 10 µg

soybean trypsin inhibitor/ml, 15 µg calpain inhibitor I and II/ml, and 5 mm NaF] for 3 min on ice.

MilliQ ice water (400 µl) was then added to swell cells for 3 min. The cells were sheared by eight

passages through a 23-gauge hypodermic needle. Nuclei were recovered by centrifugation at 400 x

g and 4C for 6 min and washed once in 400 µl washing buffer [10 mm Tris-HCl (pH 7.4) and 2 mm

MgCl2, plus protease inhibitors as describe above]. The purity of the isolated nuclei was analyzed

by detection of β-tubulin. Whole cells or the purified K562 nuclei were solubilized in MPER lysis

buffer (Pierce) containing calpain I/II inhibitors and including protease and phosphatase inhibitors

(Roche, Milan, Italy) . Nuclear or total protein (60 µg) was separated by SDS-PAGE gels,

transferred to a nitrocellulose membrane, and blotted with the following antibodies : anti-

PLCβ1(sc-9050), and anti-cyclin D3 (sc-182), γ-globin and lamin B rabbit polyclonal antibodies

Santa Cruz Biotechnology (SCBT), CA, USA (SCBT) and anti-β-actin monoclonal antibody

(Sigma). After being probed with the specific antibodies, the membranes were developed with

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SuperSignal West Pico chemiluminescent reagent (Pierce) and then visualized and analyzed in a

Kodak digital image station (2000R; Eastman Kodak, Rochester, NY, USA).

Transfections and isolation of clones

Cells were transfected with full-length DNA vectors for human PLCβ1a and PLCβ1b cloned into

pcDNA/2.1 plasmid. Overexpression of PLCβ1 isoforms was performed using Lipofectamine 2000

from Invitrogen. Cells were seeded at a cell density of 5x105/ml in 25 ml flasks, to which was

added the mix of Lipofectamine 2000 and the right vectors, following manufacturer’s instructions.

Cells were collected to be analyzed 24 h after the transfections. To obtain stable clones, cells were

selected by limited dilution in complete RPMI 1640 10% FBS containing Geneticin (G418 from

Sigma Aldrich) 24 h after the transfection, then expanded and kept always in selection with G418.

Cell viability analysis and apoptosis and flow cytometry

The sensitivity of K562 cells to kinamycnin F and apoptosis was evaluated by using the MTT (3-

[4,5-Dimethylthythiazol-2-yl]-2,5-Diphenyltetrazolium Bromide) cell-viability kit (Roche) or by

the binding of annexin V-FITC to phosphatidylserine exposed on the cell surface, using the

AnnexinV-FLUOS kit (Roche) , both according to the manufacturer’s instructions. In particular,

K562 cells (2 x 106 cells/ml) were cultured in triplicate in flat-bottomed 96-well plates at 37°C with

5% CO2. Cultures were performed for 72 h in complete medium supplemented or not with

Kinamycin F. Results were statistically analyzed using GraphPadPrism Software (GraphPad

Software Inc. version 3.2). To assess the extent of apoptosis induction after treatment with

kinamycin F, a flow cytometric analysis of Annexin V-FITC/ PI-stained samples was performed, as

reported. Samples were analyzed on an FC500 flow cytometer (Beckman Coulter) with the

appropriate software (CXP, Beckman Coulter). Apoptosis assays were performed using an Annexin

V-FITC detection kit I (BD Biosciences Pharmingen). The cells were analyzed using a FACScan

flow cytometer and data were evaluated using WinMDI 2.8 research software. The results were

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expressed as percentage of apoptotic Annexin V-FITC positive cells with respect to total cells

counted. For detection of surface antigens, cells were first stained with the FITC conjugated anti-

CD71 antibody for 15 minutes or irrelevant antibody. Cells were then washed twice in PBS

supplemented with 4% FBS and analyzed by flow cytometry.

RNA extraction, retrotranscription and real-time PCR analysis

Total cellular RNA was extracted using the RNeasy minikit (Ambion) according to the

manufacturer’s instructions. Two mg of total RNA were reverse transcribed using Moloney Murine

Leukemia Virus Reverse Transcriptase (M-MLV RT) and Oligo(dT) 15 primer in the presence of

dNTPs (Promega). Gene expression of PLCβ1a and PLCβ1b was determined by using the TaqMan

Gene Expression Master Mix and the 7300 real-time PCR system (Applied Biosystem). Results

were normalized to the level of the ubiquitously expressed RNA 18S ribosomal 1 gene (RN18S,

Hs03928990_g1). For PLCβ1b the Hs01001939 probe was used; for PLCβ1a, the Hs01008373

probe was used (Applied Biosystem).

Statistical analyses

Statistical analyses were performed by Student’s-test, using GraphPad Prism (GraphPad Software

Inc version 6)(*p<0.05, **p<0.001,***p<0.0001)

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Results

Kinamycin F decreases PLCβ1a protein levels but does not affect PLCβ1b

As we have just mentioned there are two PLCβ1 splice variants, PLCβ1a and PLCβ1b. At first, we

evaluated the expression of these two isoforms in K562 cells following treatment with the

antibacterial drug kinamycin F. To investigate the expression of PLCβ1 during K562

differentiation, cells were induced to differentiate by treatment with differentiation media

containing 2,5 or 5 µM kinamycin F for 24h. Cell lysates were performed and analyzed for the

presence of PLCβ1 (Fig.1a). Western blots were carried out using an antibody to PLCβ1 that

recognizes both PLCβ1a and -β1b. Both PLC β1a and -β1b isoforms were present in whole cell

lysates of uninduced K562 cells. The levels of PLCβ1a decreases upon differentiation, whilst the

levels of PLCβ1b do not after 24 h of differentiation, therefore the effect of the drug is mainly

directed towards the expression of PLCβ1a. Also quantitative PCR was used to examine PLCβ1a

and -β1b expression in K562 cells. Interestingly, qPCR analysis revealed a significant decrease of

PLCβ1a in differentiated erythrocytes compared to undifferentiated control cells. On the contrary

PLCβ1b expression was slightly reduced even at 5 µM of kinamycin F treatment. These data

prompt us to realize that kinamycin F acts selectively on PLCβ1a protein levels. Hemoglobin

production is the main marker of erythroid differentiation. In order to assess whether kinamycin F

affects erythroid cell differentiation, the marker specific to erythroid cell differentiation, i.e. γ-

globin, was monitored in the same samples. A significant increase in the level of γ-globin protein

expression was detected after 24 h of 2,5 and 5 µM kinamycin F treatment.

Effect of over-expression of PLCβ1a and of β1b on K562 differentiation

To further elucidate whether PLCβ1 could regulate differentiation, K562 cells were stably

transfected with pRc/CMV plasmids containing wild type (wt) PLCβ1a and -β1b. To determine the

biological activity of kinamycin F, we employed three experimental cell systems: (1) the human

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leukemic K562 cell line, (2) K562 cell clones stably transfected with a pcDNA/2.1 construct

carrying PLCβ1a and (3) K562 cell clones stably transfected with a pcDNA/2.1 construct carrying

PLCβ1b. The level of expression of the PLC β1 subtypes in undifferentiated and differentiated cells

was analyzed by Western blotting. PLC β1a and -β1b are effectively over-expressed as it is shown

in fig. 2. Moreover, in cells transfected with PLC β1a, the expression of PLCβ1a is significantly

reduced in the sample treated for 16 h with 5 µM kinamycin F while PLCβ1b is not affected. The

treatment with 5 µM kinamycin F for 24h of K562 overexpressing PLCβ1a is able to almost

completely knocked down PLCβ1a expression but the effect of the drug on K562 overexpressing

PLCβ1b is much less evident. Therefore, K562 cells treated with kinamycin F exhibited markedly

reduced levels of PLCβ1a but not of PLCβ1b. These results suggest that kinamycin F modulates

selectively PLCβ1a and -β1b expression. To further investigate the effect of overexpression of

PLCβ1a and of -β1b on erythroid differentiation, we monitored the expression of erythroid marker

γ-globin at 24h of culture under exposure to 5µM drug’s concentration. In control conditions,

without PLCβ1 overexpression, the majority of the cells became erythroid and expressed the γ-

globin protein. Exposure of K562 cells to PLCβ1a overexpression did not affect γ-globin

expression in the presence of kinamycin F, suggesting a delay in erythroid differentiation, whereas

higher expression of PLCβ1b significantly increased the expression of the erythroid marker. As

shown in Fig. 2a, the overexpression of PLCβ1b induced an increase in γ-globin expression,

compared with untreated control cells, even in the absence of kinamycin F. Our previous study

showed that cyclin D3 is a target of PLCβ1 signaling in a model of murine erythroleukemia.

Moreover kinamycin F caused a selective reduction of cyclin D3 protein mediated at the level of

transcription. Therefore we analyzed the effect of PLCβ1 overexpression on cyclin D3 under the

treatment of kinamycin F. After treatment with kinamycin for 24 h we note that the expression of

cyclin D3 in wild type cells is down regulated as we expected. But the amplification of the

expression of the PLCβ1a is able to maintain high levels of expression of cyclin D3 even after the

cells have been treated with the drug. It is interesting to note however that with regard to the cell

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clones in which was amplified the expression of PLCβ1b, there was a level of cyclin D3 expression

substantially low compared to wild type cells, even in proliferating cells. All these data prompted us

to believe that the overexpression of PLCβ1a is able to maintain the cells in a proliferative state and

to the contrary, the expression of PLCβ1b already in itself induces the differentiation process in

some way, even in the absence of treatment with kinamycin F.

The nuclear localization of PLCβ1 subtypes in differentiated K562 cells

Since the data obtained up to now show a different role of the two isozymes in the context of the

differentiation process, we proposed to determine whether there was a correspondence between the

different role of the two isoforms in regulating the expression of cyclin D3 and globin and their

distribution within the cell. We viewed the localization of the two isotypes in K562 cells. Through

an analysis of nuclear lysates of cycling cells and cells induced to differentiate with kinamycin F,

the result (fig. 3) is that both isoforms are present in the nuclear compartment of these cells, but we

found that PLCβ1b has a substantially higher nuclear localization as compared to PLCβ1a. A

similar difference in localization is also found in differentiated K562 cells where PLCβ1b is not

down regulated in respect to PLCβ1a as we already seen in whole cell lysates. These data suggested

even further a different role of the two isozymes in the kinamycin F induced differentiation process,

due to their different distribution within the cells.

Analysis of cell viability after treatment with kinamycin F

The effect of overexpression of the two isozymes on cell proliferation was further analyzed at 24 h

after kinamycin F administration. MTT assay in concentration-dependent manner showed that

kinamycin F in wild type cells considerably inhibited the proliferation of K562. As shown in Fig. 4

treatment of cells with kinamycin F at 2,5 and 5 µM concentration lowered cell proliferation. MTT

assay was conducted on wild type cells and on cells transfected with PLCβ1a or PLCβ1b. PLCβ1a

overexpressing cells showed stronger growth ability than the PLCβ1b ones and wild type cells,

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suggesting that K562 cells overexpressing PLCβ1a have drug resistance potential against kinamycin

F by means of a proliferative effect. Conversely, PLCβ1b overexpressing cells revealed remarkably

decreased growth ability compared with PLCβ1a overexpressing cells demonstrating once more

that PLCβ1a is able to maintain the cells in a proliferative state.

PLCβ1a protects K562 cells from apoptosis

To determine whether the loss of cell viability induced by kinamycin F was associated with

apoptosis, cells were then analyzed for positivity to Annexin V staining. The assay evaluates

phosphatidylserine turnover from the inner to the outer lipid layer of the plasma membrane, an

event typically associated with apoptosis. K562 cell clones overexpressing PLCβ1a have a lower

level of apoptosis compared to the wild type cells and also compared to the cells in which the

PLCβ1b is amplified (Fig. 5). This data is most noticeable at a concentration of 2.5 µM of

kinamycin F. It seems, therefore, that a low dose of the drug induces an apoptotic phenomenon

while a higher dose induces the differentiation of K562 directly. Flow-cytometric analysis of wild

type cells and cells overexpressing PLCβ1b, did not show significant differences in Annexin V

analysis demonstrating that both had the same effect on apoptosis process. Cells overexpressing

PLCβ1a, likely due to the fact that they are in a proliferative state, have a protective effect against

the phenomenon of apoptosis but show a delay in the differentiation process.

Flow cytometric analysis of the expression of CD71

We also performed flow cytometry analysis to assay expressions of the erythroid surface markers

CD71. The transferrin receptor (CD71) is an integral membrane protein that mediates the uptake of

transferrin-iron complexes. The level of transferrin receptor expression is highest in early erythroid

precursors through the intermediate normoblast phase, after which expression decreases through the

reticulocyte phase. The maturation to erythrocytes results in loss of transferrin receptor expression,

in concert with down-regulation of the machinery for hemoglobin synthesis (REF). To determine

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the effect of PLCβ1a and PLCβ1b on the process of erythroid differentiation, cells were gated for

flowcytometric analysis after incubation with anti-CD71 antibodies. The overexpression of PLCβ1a

raised the percentage of CD71+ cells, hinting at a marked delay of the erythroid differentiation of

K562 cells compared to the wild type cells and to cells overexpressing PLCβ1b. In contrast, the

overexpression of PLCβ1b significantly promoted the erythroid differentiation of K562 cells as

supported by decreased percentage of CD71+ (Fig.6). This event is amplified by treatment with the

drug, in fact this effect of Kinamycin F on CD71+ cells is amplified in PLCβ1a overexpressing

cells treated for 24 hours with 2.5 µM kinamcin F. These data confirm, once again, that an increase

of expression of the PLCβ1a is capable of maintaining the cells in a proliferative state, preventing

their entry into the differentiation program.

Discussion

Our previous investigations have shown that PLCβ1 is a key molecule for nuclear inositide

signaling and plays a role in cell cycle progression, proliferation and differentiation. In this study

we investigated the effect of kinamycin F on erythroid differentiation in the myelogenous leukemic

cell line K562. We already showed that both PLCβ1 expression levels and cellular localization are

necessary for the induction of erythroid differentiation. Moreover we previously demonstrated that

cyclin D3/cdk4 is a target of nuclear PLCβ1 signaling, which is able to activate cyclin D3 promoter

transcription during differentiation (Faenza et al., 2002; Faenza et al., 2007) . It has been recently

shown that treatment of K562 cells with kinamycin F induced erythroid differentiation and caused a

selective reduction of cyclin D3 protein, which appeared to be mediated at the level of transcription

(O'Hara et al.). By these evidences we hypothesized whether it could be that kinamycin F, or one of

its metabolites, could interact with PLCβ1 upstream of cyclin D3 transcription, altering the

abundance or activity of a specific factor or factors affecting erythroid differentiation process. At

first we evaluated the expression level of both the PLCβ1 isozymes, PLCβ1a and -b, after induction

of K562 cells to erythroid differentiation with the drug kinamycin F. Our results indicated that

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Kinamycin F was able to induce K562 cells differentiation at a very early stage in respect to other

inducers such as AraC or azacitidine or Hemin (Chen and Wu, 1994). Indeed γ-globin induction

occurs already after 16 hours of treatment with the drug. At the same time to the induction of γ-

globin expression, the expression of PLCβ1a was down-regulated and the expression of PLCβ1b

was down-regulated too even if to a lesser extent. Therefore from these results we assisted to a

different dose-dependent effect of the drug Kinamycin F on the regulation of the two PLCβ1

isozymes expression. Thus, to understand the possible role of the two PLCβ1 isoforms we decided

to make use of two experimental models. We constructed stable clones of K562 cells in which

PLCβ1a and PLCβ1b expression level was respectively amplified. K562 cells treated with

kinamycin F exhibited markedly reduced levels of PLCβ1a but not of PLCβ1b. These results

suggested that kinamycin F modulates selectively PLCβ1a and -1b expression. Experiments were

conducted to assess whether PLCβ1 signaling pathway was involved in the regulation of cyclin D3

expression after kinamycin F treatment. All the experiments that we carried out have shown

unequivocally that the amplification of the PLCβ1a is able to keep the cells proliferating, instead of

the amplification of PLCβ1b which somehow induce already in itself the differentiation process

even in the absence of the drug. This is evident from the fact that amplification of PLCβ1a

expression is able to maintain high levels of cyclin D3 levels even after the cells have been treated

with the drug. Moreover exposure of K562 cells to PLCβ1a overexpression did not affect γ-globin

expression in the presence of kinamycin F, suggesting a delay in erythroid differentiation, whereas

higher expression of PLCβ1b significantly increased the expression of this erythroid marker. We

have previously reported that nuclear localization is crucial for the function of PLCβ1 but the data

obtained in this study show and demonstrate, for the first time, an extremely different role of the

two isozymes in the processes of proliferation and differentiation as well. Subsequent experiments

have shown that not only the expression of the two isoforms is differently regulated by treatment

with kinamycin F but also that they are differently distributed in the cell compartments since

PLCβ1b is localized mainly in the nucleus compared to PLCβ1a. Therefore it has become

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increasingly clear that the proliferation or differentiation processes through the control of cyclin D3

expression could also be due to a different distribution of the two enzymes within the cell. Further

analysis indicate that PLCβ1a expression amplification, following treatment with kinamycin F, not

only confers a real advantage to K562 cells viability but also that it protects cells themselves from

apoptosis. In the final step, to determine the effect of PLCβ1a and PLCβ1b on the process of

erythroid differentiation, we also examined the level of transferrin receptor expression. Our results

demonstrate that PLCβ1a and PLCβ1 b have different roles in that the overexpression of PLCβ1b

significantly promoted the erythroid differentiation of K562 cells as supported by decreased

percentage of CD71+. Overall, in this study we showed that during K562 differentiation process

induced by kinamycin F, cyclin D3 level is regulated by PLCβ1 signaling pathway. We highlighted

that PLCβ1a and PLCβ1b are differently localized within the cell being PLCβ1b mainly present in

the nucleus in respect to PLCβ1a. We assessed the different action of Kinamycin F in the regulation

of PLCβ1a and -1b expression due to their different localization. As previously shown PLCβ1

inhibits, in human K562 cells, erythroid differentiation induced by mithramycin by targeting miR-

210 expression and exerts an impairment of normal erythropoiesis as assessed by γ-globin

expression (Bavelloni et al., 2014). Another study on the effects of the overexpression of PLCβ1

showed a specific and positive connection between cyclin D3 and PLCβ1 in K562 cells, which led

to a prolonged S phase of the cell cycle and a delay in cell proliferation in that PLCβ1 targets cyclin

D3, likely through a PKCα-mediated-pathway and, as a downstream effect of its activity, K562

cells undergo an accumulation in the S phase of the cell cycle (Poli et al., 2013). The major

regulators of PI4,5P2-mediated events are PIP kinases, which in turn are involved in PI4,5P2

synthesis (York et al., 1999) (Clarke et al., 2010) (Shah et al., 2013). Very recently De

Albuquerque Wobeto et al. show that PIPKIIa is widely expressed in hematopoietic derived cells, is

localized in the cytoplasm and nucleus, and is upregulated during erythroid differentiation of K562

cells. PIPKIIa silencing resulted in an increase in the expression of α and γ globins and a slight

decrease in cell proliferation in K562 cells without any effect on the modulation of cell cycle

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progression and apoptosis. The change in nuclear Polyphosphoinositides in response to stimuli

indicated a specific signalling role for Polyphosphoinositides within the nucleus. Through direct

interaction with downstream effectors or generation of Ins(1,4,5)P3 and higher phosphorylated

inositols and interaction with their effectors, the signals are transduced into changes in cellular

pathways. The number of potential effectors has dramatically increased as a consequence of global

proteomic studies to identify nuclear proteins that may interact with Polyphosphoinositides and

PLCβ1(Lewis et al., 2011) (Piazzi et al., 2013). All in all we have identified a different role for the

two isozymes of PLCβ1 as regulators of K562 differentiation process induced by Kinamycin F.

PLCβ1a resides predominantly at the plasma membrane where it is associated with Gαq. In

addition, PLCβ1a can be found in the cytosol and the nucleus (Follo et al., 2014). Nuclear PLCβ1 is

the physiological target of ERK. In response to IGF-I stimulation, the activated ERK in the nucleus

phosphorylates PLCβ1 at serine 982, which is located in the characteristic long carboxyl-terminal

domain that has been shown to possess a number of regulatory functions (Xu et al., 2001). A less

direct, but very intriguing, link between PLCβ1 and differentiation comes from the finding that one

allele of this enzyme is deleted in a subset of high risk myelodysplastic syndrome (MDS) patients,

that show an unpredictable aggressive disease course and develop acute myeloid leukemia in a

shorter time frame, in comparison to MDS patients whose alleles for PLCβ1 are both intact (Cocco

et al., 2005). Moreover both the level of expression and the level of methylation of PLCβ1

promoter are good prognostic indicators in demetilating therapy of MDS patients (Follo et al., 2012)

(Follo et al., 2009) (Follo et al., 2013). Nuclear PLCβ1a is implicated in the progression of MDS to

AML, by genetic and epigenetic mechanisms. The present study demonstrates that following

treatment with kinamycin F, PLCβ1a and – β1b are relevant regulators of erythropoiesis, having

opposite effect such as a positive or a negative function in erythroid maturation, on the basis of their

intracellular localization.

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Acknowledgments

This work was supported by the Italian MUIR-FIRB 2010 Accordi di Programma (to LC), by the “5

per 1000(2011–2012)” fund to the SC Laboratory of Musculoskeletal Cell Biology, Rizzoli

Orthopedic Institute and by a Discovery grant from the Natural Sciences and Engineering Research

Council of Canada (to GID).

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Figure legend

Fig.1 Levels of PLCβ1, in control cells and in cells exposed to Kinamycin F administration (2,5

and 5 µM). a): Whole-cell lysates were prepared and 80 µg was loaded in each lane for separation

by SDS-PAGE on 8% acrylamide gels and then analyzed with specific antibodies. One of the

membranes was reprobed with β-actin antibody to normalize the amount of loaded proteins. Cells

were then lysed to extract RNA and proteins. Western blotting analyses were carried out with

specific antibodies against PLCβ1 and β-actin. γ-globin expression was used to check the

differentiation of K562 cells. b): The extracted RNA was retro-transcribed and real-time PCR was

performed to evaluate the quantitative expression of PLCβ1a, PLCβ1b.

Fig. 2 Effect of PLCβ1 overexpression on the differentiation of K562 cells. a) K562 cells were

stably transfected with PLCβ1a or PLCβ1b. After 16 or 24 h of kinamycin F administration (2,5 or

5 µM), whole homogenates were separated by SDS-PAGE and analyzed by immunoblot with

specific antibodies. One of the membranes was reprobed with an anti-β-actin antibody to verify

equal amounts of loaded protein (bottom panel). b): Stably transfected K562 cells were

differentiated and the level of differentiation was analyzed by γ-globin expression in the presence

and in the absence of 5µM kinamycin F (24h). One of the membranes was reprobed with anti-cyclin

D3 antibody. Results are representative of 3 independent experiments.

Fig. 3 Analysis of PLCβ1 phenotype in K562 cells wt. Nuclear lysates (80 µg of protein/lane) were

resolved by SDS–6% PAGE and then analyzed by immunoblotting with antibodies against the

PLCβ1a and β1b. One of the membrane was reprobed with anti-lamin-B antibody and with anti-β-

actin antibody to verify equal amounts of loaded protein and the purity of nuclear extracts.

Fig.4 MTT assay of K562 control cells and cells transfected with PLCβ1a or PLCβ1b. Cells were

treated with different concentration of kinamycin F. The mean of 3 different experiments sd is

plotted. Statistically significant differences were determined by 2-way ANOVA.***P 0.0001;

significant with respect to both the mutation and the time.

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Fig. 5 PLCβ1a inhibits apoptosis in K562 cells. Apoptosis assessment by Annexin V/propidium

iodide staining shows the apoptotic rates of K562 cells transfected with PLCβ1a or PLCβ1b and

treated with kinamycin F (2,5 and 5 µM) for 24 h. The data are representative of 3 separate

experiments.

Fig.6 The overexpression of PLCβ1b significatively promotes the erythroid differentiation of K562

cells. Flow-cytometric analysis was performed on K562 control cells and cells transfected with

PLCβ1a or PLCβ1b. Cells were treated with kinamycin F for 24 h (2,5 and 5µM) and then labeled

with antibodies directed against CD71. The data are representative of 3 separate experiments.

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