1. Introduction Conclusions
PLC1 regulates the expression of Cyclin D3 via PKCa leading to a prolongation of the
S phase of the cell cycle in human erythroleukemia cells (k562)
Alessandro Poli*, Irene Faenza*, Alessandro Matteucci*, Francesca Chiarini*, Lucio Cocco*
*Cellular Signaling Laboratory, Department of Human Anatomy, University of Bologna, Italy
Introduction
PLCb1 is one of the most important protein of nuclear cell signalling. It is already known that it
is present in two different isoforms, PLCb1a (more cytoplasmatic) and PLCb1b
(mostly nuclear). Our previous works demonstrated PLCb1 to be involved in cell cycle control
during the G1/S transition targeting Cyclin D3 in MEL cells. We used human erythroleukemia cells (K562)
to investigate the presence of this link in human cells and to further study how this mechanism could work
Fig.1 Transient PLCb1 overxepression increases Cyclin D3 levels and decreases PKCa expression; this regulation leads to a prolonged S phase of the cell cycle and a subsequent slower proliferation
A) Starved
24h
B) St + 6h
10% RPMI
C) St + 24h
10% RPMI
A) Starved 24h B) St 24h C) St 24h +
RPMI 10% 6h RPMI 10% 24h
D) Proliferation
Ctrl OV
Ctrl OV
Ctrl OV
PLCb1
PKCb1
PKCa
P-PKCa
Cyclin D3
b-tubulin
Fig.1 Cells were grown in RPMI 10% FBS and transiently transfected with PLCb1 or empty vector
A) Cells starved for 24h with RPMI without FBS;
B) Cells starved for 24h with RPMI without FBS and resuspended in complete RPMI 10% for 6h;
C) Cells starved for 24h with RPMI without FBS and resuspended in complete RPMI 10% for 24h;
D) Cells seeded at a density of /ml and counted at h to check the proliferation
Fig.2 Stable overexpression of both the isoforms of PLCb1 (1a and 1b) shows an higher Cyclin D3 expression, a prolonged S phase of the cell cycle
and a slower proliferation than the control; PKCa expression is lower with OV of PLCb1a/1b than the control
A) Starved
24h
B) St+ 6h
RPMI 10%
C) St+24h
RPMI 10%
C) 24h starvation + 24h RPMI 10%
D) Proliferation
Ctrl OV1b OV1a
Ctrl OV1b OV1a
Ctrl OV1b OV1a
PLCb1
PKCa
Cyclin D3
b-tubulin
Fig.2 Cells were stably transfected with PLCb1a, PLCb1b and empty vectors; clones were selected via G418
A) Clones starved for 24h with RPMI without FBS;
B) Clones starved for 24h with RPMI without FBS and resuspended in complete RPMI 10% for 6h;
C) Clones starved for 24h with RPMI without FBS and resuspended in complete RPMI 10% for 24h;
D) Clones seeded at a density of /ml and counted at h to check the proliferation
Fig3. PKCa knock-down increases Cyclin D3 levels via the decrease of p27 and leads to a slower proliferation than the scrambled sample
A) Transfection
PKCa Kd Scr
PKCa Kd Scr
B) Proliferation
PLCb1
PKCa
KD Scr KD Scr KD Scr
24h h h
PKCa
Cyclin D3
PKCa
P-PKCa
p27
b-tubulin
b-tubulin
b-tubulin
Fig.3 K562 transiently transfected with siRNAs to knock-down PKCa and scrambled as control
A) Cells 24h after transfection;
B) Cells were seeded at a density of /ml and, after transfection, counted for three days
Conclusions
The presented data confirm the role of PLCb1 in cell cycle regulation also in human erytrholeukemia cells (K562). Our work suggests a possible inverse correlation between PLCb1 and PKCa that could lead to a regulation of Cyclin D3 via p27 and then to a prolonged S phase of the cell cycle
PLCb PKCa p Cyclin D3
Prolonged S phase of the cell cycle
References
1) A Role for Nuclear Phospholipase Cb1 in Cell Cycle Control
Irene Faenza, Alessandro Matteucci, Lucia Manzoli, Anna Maria Billi, Michela Aluigi, Daniela Peruzzi, Marco Vitale, Sergio Castorina, Pann-Ghill Suh and Lucio Cocco
2) PKC alpha affects cell cycle progression and proliferation in human RPE cells through the downregulation of p27kip1
Qianying Gao, Juan Tan, Ping Ma, Jian Ge, Yaqin Liu, Xuerong Sun, Lian Zhou State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
3) Protein Kinase C alpha-mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor Induced Activation of Nuclear Phospholipase C b1 in Swiss 3T3 Cells*
Aimin Xu, Yu Wang, Lance Yi Xu, and R. Stewart Gilmour
From the ‡Liggins Institute, School of Medicine, and §School of Biological Sciences, University of Auckland,Private Bag 92019, Auckland, New Zealand