lysophosphatidic acid induces upregulation of mcl-1 and protects

8/22/2019 Lysophosphatidic Acid Induces Upregulation of Mcl-1 and Protects

1/15

Nguyn Tng Trng

[email protected]

Lysophosphatidic acid induces upregulation of

Mcl-1 and protects apoptosis in a

PTX-dependent manner in H19-7 cells


2/15

A B S T R A C T Lysophosphatidic acid (LPA) is a lipid growth factor known to regulate diverse cell

functions, including cell proliferation, survival, and apoptosis. Tight regulation of cellsurvival in neuronal precursor is essential during neurogenesis in both developingand adult brain. Increasing data show that diverse external factors including LPA play

roles in controlling cell survival and apoptosis in early developing neurons./However,the underlying control mechanism remains unclear./ To explore how LPA regulatescell survival or apoptosis in a developing neuron, mechanisms for cell survival and

signaling cascades by LPA were investigated in H19-7 hippocampal progenitor cells./Here, we showed that LPA promotes cell survival by protection from apoptosis. Mcl-1was demonstrated to be crucial in LPA-induced cell survival by transfection of thesiRNA specific for Mcl-1 and overexpression of Mcl-1. LPA-induced cell survival wascritically mediated by the upregulation of Mcl-1 which was regulated not onlythrough a post-translational control but a transcriptional control. Mcl-1 stabilization byLPA-induced inhibitory phosphorylation of GSK-3 contributed predominantly to theMcl-1 upregulation. Both LPA-induced cell survival and the GSK-3 phosphorylation

were attenuated by PTX and by siRNA specific for LPA1 or LPA2 receptor./ Takentogether, these results showed that Mcl-1 stabilization by inhibitory phosphorylation ofGSK-3 through Gi/o coupling of the LPA1 and LPA2 receptors following Mcl-1

upregulation plays a critical role in LPA-induced survival of H19-7 cells./ Indeveloping neurons, modulation of Mcl-1 levels may constitute a crucial mechanismfor controlling their fates.

Backgrounds

Questions

Experiments

Results

Answers

Speculations


3/15

1/ABSTRACT Original: Mcl-1 stabilization

by LPA-induced inhibitoryphosphorylation ofGSK-3contributed predominantlyto the Mcl-1 upregulation.

My suggestion: Mcl-1stabilization by LPA-induced inhibitoryphosphorylation of

Glycogen synthasekinase 3 (GSK-3)contributed predominantlyto the Mcl-1 upregulation.

Keywords:

Lysophosphatidic acid

Neuronal progenitor cell

Neurogenesis Mcl-1

Glycogen synthase kinase 3

Cell survival


4/15

A B S T R A C T Here, we showed that LPA promotes cell survival by

protection from apoptosis. Mcl-1 was demonstrated to becrucial in LPA-induced cell survival by transfection of thesiRNA specific for Mcl-1 and overexpression of Mcl-1. LPA-induced cell survival was critically mediated by theupregulation of Mcl-1 which was regulated not only througha post-translational control but a transcriptional control. Mcl-1stabilization by LPA-induced inhibitory phosphorylation ofGlycogen synthase kinase 3 (GSK-3)contributedpredominantly to the Mcl-1 upregulation. Both LPA-inducedcell survival and the GSK-3 phosphorylation were attenuatedby Pertussis toxin (PTX) and by siRNA specific for LPA1 orLPA2 receptor./

Taken together, these results showed that Mcl-1 stabilizationby inhibitory phosphorylation of GSK-3 through Gi/o couplingof the LPA1 and LPA2 receptors following Mcl-1 upregulationplays a critical role in LPA-induced survival of H19-7 cells./

In developing neurons, modulation of Mcl-1 levels mayconstitute a crucial mechanism for controlling their fates.

Results

Answers

Speculations


5/15

INTRODUCTION

LPA, a phospholipid growth factor,

affects various cell functions,

including cell proliferation,

differentiation, survival, and

migration through a family of

cognate G-protein coupledreceptors (GPCRs). Binding of

LPA to its cognate receptors elicits

a variety of LPA down- stream

signaling through at least three

different types of G proteins,

including...

TOPIC SENTENCE


6/15

INTRODUCTION including Gi/o, Gq, and G12/13 [1]. Six different LPA receptors have beenidentified, whose expression varied in different tissues. Differentialexpression patterns of LPA receptors in neuronal cells during many

neurodevelopmental processes, including neurogenesis, neuritogen- esis,

and neuronal migration suggest that LPA may play roles in neuronal

development. In fact it was shown that targeted deletion of lpA1 resulted in

increased apoptosis in sciatic nerve Schwann cells and developmentalabnormalities in olfactory bulb and/or cerebral cortex [2]. In addition LPA

produced gyri-like cortical folds by promoting cell survival of neuronal

progenitor cells (NPC) within the cerebral cortex in ex vivo cortical culture

system [3]. However, molecular mechanisms by which LPA regulates thesurvival of NPC are obscure. Even though LPA is shown to play roles in

regulating cell survival in diverse neuronal cells during development, the

effects of LPA on the neuronal cell are controversial and their detailed

mechanism remains to be elucidated. LPA promoted the survival of

cultured hippocampal neurons from embryonic day 16-17 ( E16-17) rats

[4,5], whereas LPA stimulated apoptosis in hippocampal neurons from E18

[6,7]. LPA promoted the survival of NPC within the cerebral cortex in ex

vivo culture of E14 cortices [3], while LPA stimulated moderately cell

proliferation in cortical blast cells from E12-13 cerebral cortex [2].

Although diverse signaling mechanisms that control LPA-induced cell

survival have been suggested [8], the mechanism for LPA-induced cell

survival in a developing neuron may not be explained simply by the

results based on previous observations for LPA-induced cell survival or

apoptosis in diverse nonneuronal cells or even in adult neuronal cells due

to complicated differential apoptotic mechanisms in a developing neuron.

To clarify these controversies and to investigate how LPA controls cell

survival or apoptosis in a developing neuron, we examined the effect of

LPA on cell viability and delineated the mechanism by which LPAregulated cell viability in H19-7 cells, an immortalized hippocampal

progenitor cell line.

In the present study, we demonstrated that LPA protects H19-7 cells

against apoptosis by upregulation of Mcl-1 predominantly through a post-

translational control via inhibitory serine phosphorylation of GSK-3.

Contrary to tyrosine phosphorylation of GSK-3 through G12/13 leading tocell death [9], serine phosphorylation of GSK-3 through

Gi/o resulted in cell survival in neuronal cells expressing mcl-1. In a

developing brain highly sensitive modulation of Mcl-1 levels by

regulation of GSK-3 activity through not only neurotrophic factors but

GPCR agonists including LPA may play a crucial role in con- trollingthe survival fate of NPC during neurogenesis in the cortex andhippocampus.

OVERALL CONCLUSIONS

GAP IN EXISTING KNOWLEDGE

BACKGROUND OF STUDY

PREVIOUSSTUDIES


7/15

2. MATERIALS AND METHODS 2.1. Materials LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate), fatty acid-free bovine serum albumin (BSA), G418,

pertussis toxin (PTX), and LiCl were purchased from Sigma (St. Louis, MO). Antibody specific to phospho-histone H3Ser10, TOP Flash, and FOP Flash luciferase reporters were purchased from Upstate Biotechnology(Lake Placid, NY). Antibody against phospho-specific p38 mitogen-activated protein kinase (MAPK),extracellular signal-regulated kinase (ERK), cAMP response element binding protein (CREB), glycogensynthase kinase (GSK)-3Ser21, GSK-3Ser9 and -actin were obtained from Cell Signaling Technology(Beverly, MA). Antibody specific to phosho- GSK-3tyr279/tyr216 was purchase from Biosource (SanJose, CA). Antibodies to Mcl-1, Bcl-xL, and Bcl-2 were purchased from Santa Cruz Biotechnology (Santa Cruz,CA). Alexa Flour 568-conjugated goat anti-rabbit IgG and Alexa Flour 488-conjugated goat anti-mouse IgGwere purchased form Molecular Probe (Eugene, OR). PD98059, SB203580, wortmannin, and Rp-cAMP wereobtained from Tocris (Bristol, UK). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum(FBS) were obtained from Gibco/BRL (Grand Island, NY). All other reagents were of analytical grade or of thehighest purity available.

2.2. Cell culture H19-7 cells, conditionally immortalized E17 rat hippocampal cells with a temperature-sensitive SV40 large T

antigen [10], were maintained at 33 C, the permissive temperature at which SV40 large T is functional, inDMEM supplemented with 10% FBS, 0.2 mg/ml G418, 50 U/ml penicillin and 50 g/ml streptomycin. After 1day of cultivation in DMEM supplemented with N2 (N2 media) at 33 C, the cells were transferred to 39 C toeliminate expression of functional SV40 large T antigen, cultured further in N2 medium for 24 h, and treatedwith LPA as previously described [11].

be in conflict

be in conflict


8/15

2. MATERIALS AND METHODS 2.1. Reagents Prepairation LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate), fatty acid-free bovine serum albumin (BSA), G418,

pertussis toxin (PTX), and LiCl were purchased from Sigma (St. Louis, MO). Antibody specific to phospho-histone H3Ser10, TOP Flash, and FOP Flash luciferase reporters were purchased from Upstate Biotechnology(Lake Placid, NY). Antibody against phospho-specific p38 mitogen-activated protein kinase (MAPK),extracellular signal-regulated kinase (ERK), cAMP response element binding protein (CREB), glycogensynthase kinase (GSK)-3Ser21, GSK-3Ser9 and -actin were obtained from Cell Signaling Technology(Beverly, MA). Antibody specific to phosho- GSK-3tyr279/tyr216 was purchase from Biosource (SanJose, CA). Antibodies to Mcl-1, Bcl-xL, and Bcl-2 were purchased from Santa Cruz Biotechnology (Santa Cruz,CA). Alexa Flour 568-conjugated goat anti-rabbit IgG and Alexa Flour 488-conjugated goat anti-mouse IgGwere purchased form Molecular Probe (Eugene, OR). PD98059, SB203580, wortmannin, and Rp-cAMP wereobtained from Tocris (Bristol, UK). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum(FBS) were obtained from Gibco/BRL (Grand Island, NY). All other reagents were of analytical grade or of thehighest purity available.

2.2. Cell culture H19-7 cells, conditionally immortalized E17 rat hippocampal cells with a temperature-sensitive SV40 large T

antigen [10], were maintained at 33 C, the permissive temperature at which SV40 large T is functional, inDMEM supplemented with 10% FBS, 0.2 mg/ml G418, 50 U/ml penicillin and 50 g/ml streptomycin. After 1day of cultivation in DMEM supplemented with N2 (N2 media) at 33 C, the cells were transferred to 39 C toeliminate expression of functional SV40 large T antigen, cultured further in N2 medium for 24 h, and treatedwith LPA as previously described [11].

be in conflict

be in conflict


9/15

2. MATERIALS AND METHODS 2.5. Measurement of apoptosis by

annexin V staining

Apoptosis was measured by staining with

Vybrant Apoptosis Assay Kit (Molecular Probes,

Eugene, OR). H19-7 cells were collected,

washed with phosphate-buffered saline (PBS),and resuspended in

100 l of binding buffer (10 mM HEPES,

pH7.4, 140 mM NaCl, and

2.5 mM CaCl2). The cells were then incubated

with 5 l of annexin

V-Alexa Fluor 488 and 1 l of 100 g/ml of

propidium iodide (PI) for

15 min at room temperature in the dark, followed

by the addition of

400 l of binding buffer. The cells were

analyzed by flow cytometry using a GuavaEasyCyte (GE Healthcare).

Notnecessary

2.5. Apoptosis Measurement


10/15

2. MATERIALS AND METHODS 2.9. RNA interference by siRNA

and stable cell

H19-7 cells were transiently transfected withthe Dicer-substrate siRNA oligonucleotidesby using Lipofectamine 2000 (Invitrogen).The most effective siRNA oligonucleotide

directed toward each target gene waschosen among the TriFECTa siRNAs(Integrated DNA Technolo- gies) by testingthe efficacy to reduce the levels of specificmRNA.

Stable H19-7 cells expressing A-CREB, adominant negative CREB, were establishedby transfection with the pRc-CMV500which was kindly provided by Dr. David D.

Ginty (Johns Hopkins Univ., USA). Clonesexpressing A-CREB were selected bycultivating the cells in the presence ofgeneticin (600 g/ml) for 3 to 4 weeks.Expression of A-CREB in the clones wasconfirmed by the attenuation of CRE-drivenluciferase activation by LPA.

Notnecessary

2.9. RNA interference


11/15

3/RESULTS - THE FONT IN FIG. 1.


12/15

4/DISCUSSION Change the style, move up

Fig. 8.

Put Fig. 8 in the Parentasis -Backets (Fig. 8)

Regardless of themechanisms for the selectivityin coupling of GPCR to Gprotein and recruiting of GSK-3 upstream kinases to GSK-3,

our data suggests activationof PKC and probably p38MAPK, but not activation ofERK, PKA and PKB, which isfollowed by coupling of LPA1and LPA2 to Gi/o lead

effectively to inactivation ofGSK-3 in H19-7 cells asshown in Fig. 8, whilecoupling of LPA4 and LPA5to Gs can activate PKA-CREBby increasing the cAMP

LPA1 and LPA2 to Gi/o lead

effectively to inactivation of GSK-3

in H19-7 cells as shown in (Fig.

8), while coupling of LPA4 andLPA5 to Gs can activate PKA-

CREB by increasing the cAMP


13/15

5/ ACKNOWLEGMENTS Original: None Title of Authors

researches.

My suggestion: Insert the Titlesto help readers.

.

[6] F.W. Holtsberg, M.R. Steiner,J.N. Keller, R.J. Mark, M.P. Mattson,S.M. Steiner, J. Neurochem. 70 (1)(1998) 66.

[7] M.R. Steiner, F.W. Holtsberg, J.N.Keller, M.P. Mattson, S.M. Steiner,

Ann. N.Y. Acad. Sci. 905 (2000) 132. [8] J. Radeff-Huang, T.M. Seasholtz,

R.G. Matteo, J.H. Brown, J. Cell.Biochem. 92 (2) (2004) 949.

[9] C.L. Sayas, J. Avila, F.Wandosell, J. Neurosci. 22 (16)(2002) 6863.

[10] E.M. Eves, L.H. Boise, C.B.Thompson, A.J. Wagner, N. Hay,M.R. Rosner, J. Neurochem.

67 (5) (1996) 1908.

Wheres my Titles ?

Whats my research ?

Scientific research articles provide a method for scientists to

communicate with other scientists about the results oftheir research.


14/15

6/ TILTLE OF ARTICLE Original: Lysophosphatidic acid induces upregulation of Mcl-1 and

protects apoptosis in a PTX-dependent manner in H19-7 cells

My suggestion:1/Crucial role of lysophosphatidic acid in apoptosis protection

in H19-7 cells by control the restriction of Mcl-1.

2/Lysophosphatidic acid in apoptosis protection in H19-7 cells

by control the restriction of Mcl-1.

3/Lysophosphatidic acid regulates NPC survival by controlthe restriction of Mcl-1.

4/Influences of Lysophosphatidic acid to NPC survival by

apoptosis protection, control the restriction of Mcl-1 in

H19-7 cells.


15/15

QUESTIONS

Rows in Abstract, Rows Introduction.

How many Sentences ?

Positions of Fig. (top or bottom)

Fantastic Class and Fantastic Teacher

!!!

lysophosphatidic acid induces upregulation of mcl-1 and protects

Documents