Chapter-II Signaling pathways involved in resveratrol …shodhganga.inflibnet.ac.in › bitstream...

Chapter-II

Signaling pathways involved in resveratrol induced neuronal differentiation in PC12 cells

Executive summary

Introduction

Materials and Method

Results

Discussion

Signaling pathways involved in Resveratrol induced neuronal differentiation

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Executive Summary

Resveratrol (RV), a polyphenol found in red wine, has been reported as an anti-aging

and antioxidant compound. It is widely consumed as a nutritional supplement, but its

mechanism of action remains an enigma. We investigate the signaling pathways and bio-

molecules associated with the RV induced neurite outgrowth in PC12 cells in presence

and absence of NGF. Our study revealed that RV induced neurite outgrowth involved

the AC1/cAMP/ Rap1-dependent pathway that leads to phosphorylation of ERK1/2 and

p38 MAP kinases. Our results also indicated that these changes are mediated through

intracellular calcium [Ca2+

]i. The ERK1/2 and p38 have also been found to play a key

role in RV induced neurite outgrowth in PC12 cells. Though, the physiological

dynamics was different in NGF and RV induced neurite outgrowth in PC12 cells and the

magnitude of differentiation was comparatively less in case of RV than NGF but, RV

was found to have a supportive role in enhancing the differentiation potential of NGF in

PC12 cells. These findings also indicate that RV activated the ERKs/p38 via the small

G-protein, Rap1 via Rap1/B-Raf signaling complex. RV was also found to induce

cAMP/Rap1-dependent pathway that leads to CREB phosphorylation via ERK1/2 and

p38 MAP kinases dependent pathway. RV was found to potentiate the action of NGF to

induce the neurite outgrowth in PC12 cells via induction of cAMP, [Ca2+

]i levels and

Rap1 signaling bio-molecules. Given the fact that these signaling molecules are highly

expressed in the central nervous system, we suggest that this signaling pathway may

regulate a number of activity-dependent neuronal functions.


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Introduction

Neurogenesis is a complex multistep process that involves cell proliferation, migration

and differentiation. The rate of neurogenesis is regulated not only by selective

expression and repression of a series of genes in neural progenitor cells at specific stages

of development, but is also affected by variety of factors like age, growth factors,

hormones, environmental or pharmacological stimuli and intercellular communications

(Vaudry, et al., 2002). It is documented that the impaired neurogenesis may be involve

in the pathophysiology of various brain diseases such as depression, epilepsy, ischemic

stroke, etc (Abdipranoto, et al., 2008; Taupin, et al., 2008). In addition to this,

environmental toxicants are also known to alter rate of neurogenesis and is result in

various pathophysiological conditions such as Alzheimer, Parkinson and Huntington

diseases. Neurogenesis is known to be driven by the flux of a variety of neurotrophic

growth factors especially Nerve growth factor (NGF). NGF belongs to the neurotrophin

family and is known to stimulate growth and differentiation of neurons during

development (Bothwell, 1995). Further NGF and its receptors have also been reported to

be elevated in activated neurons, microglia, and astrocytes. Recent research had reported

that endogenous production of NGF gradually decreases with aging in humans and its

reduction may participate in neuro degenerative diseases. It is suggested that after brain

injury, NGF serves to protect injured neurons from neuronal damage and also affect

growth of damaged axons (Sofroniew et al., 2001). Indeed, studies had proved that the

exogenous delivery of NGF induces growth of injured axons (Oudega and Hagg, 1996;

Thuret et al., 2006). However, a practical drawback to the clinical use of NGF


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supplementation as a therapeutic intervention is the fact that NGF does not readily cross

the blood-brain barrier, making its use dependent on invasive neurological interventions,

that it requires direct infusion of the neurotrophin into the cerebro-ventricular system

(Brinton and Yamazaki, 1998). In addition, because of the diversity of cell types that

respond to NGF both peripherally and centrally, NGF supplementation has led to a

number of undesirable side effects in both animals and patients, limiting the

effectiveness of its use as a therapeutic intervention to treat brain injury (Jonhagen,

2000; Winkler et al., 2000). It is important to identify biological modulators that can be

helpful in enhancement of the neuritogenic ability of endogenous NGF via

administration of exogenous potentiators. This approach would be without the systemic

interventions and can be effected the exogenous NGF administration. Several natural

products are known to directly induce neurite outgrowth or potentiate the action of NGF,

which may be suited for the treatment of neuronal injury and are without the logistical

drawbacks and high costs of synthetic drug development (Sagara et al., 2004; Tohda et

al., 2005; Kano et al., 2008; Shibata et al., 2008). Dasgupta (2007) for the first time

showed the neurite outgrowth capability of RV in neura 2 cells (mouse neuronal cells).

This study provides evidences for the promising use of RV in neuronal diffentiation

based therapies. However, still the mechanistic aspect of this property of RV is elusive.

Keeping this in mind, we hereby showed the neuronal differentiation properties of RV,

along with its capability to enhance the differentiation capability of NGF in combination

per se. The study is also quite unique in terms of model selection, as PC12 cells are non-

neuronal origin cells but in presence of NGF, they are known to differentiate into

neuronal cells. The neurite outgrowth properties of RV serve and provide clues for its


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possible use in neuro-degenerative diseases. It is for the first time, we have

demonstrated the proactive role of RV in the neuronal differentiation via induction of

intracellular calcium and by the triggering of the AC/cAMP/Rap1/ERK1/2 and p38

pathways.


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Materials and Method

Cell culture:

PC12, a rat pheochromocytoma cell line was originally procured from National Centre

for Cell Science, Pune, India, since then it has been maintained at In Vitro Toxicology

Laboratory, Indian Institute of Toxicology Research, Lucknow, India, as per the

standard protocols described. In brief, Cells were maintained in Nutrient Mixture (F-12

Hams), 82.5% supplemented with 2.5% heat inactivated fetal bovine serum (FBS), 15%

heat inactivated horse serum (HS), 0.2% sodium bicarbonate (NaHCO3), antibiotic and

antimycotic solution (10X, 1 ml/100 ml of medium, Invitrogen, Life Technologies,

USA). Cultures were grown at 37oC in incubator containing 5% CO2, an atmosphere of

95% air under high humidity. The Culture medium was replaced twice in a week and

cell cultures were passaged at a ratio of 1:6 in a week. Prior to using in the experiments,

cells were screened for integrity of established markers (Greene, et al., 1976; Galbiati, et

al., 1998) and viability (Pant, et al., 2001). Batches showing more than 95% cell

viability were used in the study. Depending upon the endpoints, cells were grown in T-

25 cm2, T-75 cm

2 flasks, 6, 12, 24, 48 and 96 well culture plates.

Identification of up & downstream regulator molecules activation in presence of

RV:

The activation of various MAP kinases is governed via different upstream molecules and

their phosphorylation has been studied. Furthermore studies were also carried out to

investigate the downstream molecules after MAP kinases activation. After exposure

with RV in presence and absence of NGF, cells were incubated for different time period.


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Analysis of upstream & downstream regulator molecules were studied through in-silico

experiments, western blotting and by commercially available kits. Moreover, various

pharmacological inhibitors such as MEK1/2 Inhibitor U0126 (10 µM), ERK1/2 inhibitor

PD 98059(20 µM), p38 inhibitor SB 203580 (10 µM), cAMP Inhibitor cAMP-Rp (15

µM), PKA inhibitor H89 (15 µM), Rap1 inhibitor GGTI 298 (5 µM) and calcium

cheletor BAPTA (10µM) were used to dissect the signaling cascade involved by RV.

In-silico experiments:

The docking studies were performed using Glide 5.6 (Glide, et al., 2010). The ligand

RV was prepared by LigPep 2.3 application using OPLS 2005 force field. The adenylate

cyclase protein (PDB Id: 1WWW) was prepared by protein preparation wizard available

in Schrodinger software package (Schrodinger, et al., 2005). The co-crystallized ligand

forskolin (FK) was selected for generation of docking grid in Glide5.6docking software

and RV was docked into the prepared grid (active site) using the automated ‘Extra

precision" (XP) mode of Glide5.6 to ensure the correct binding mode of the RV.

Measurement of cyclic-AMP levels in PC12 cells:

PC12 cells were seeded on poly-L-lysine-coated 6-well plates in normal medium for 24

h. The cells were then shifted to low serum (1% HS and 0.5% FBS) for 24 h and

exposure to RV (10µM) in presence and absence of NGF (50ng/mL) for different time

periods. Cells were treated with 0.1 M HCl after removing the culture media and

incubated for 20 min and scraped with a cell scraper. The cell lysates were centrifuged at

top speed for 10 min and the supernatant was used directly in the assay. The intracellular


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cyclic-AMP level was measured by the cAMP Direct Immunoassay Kit (Catalog no.

ab65355, Abcam, UK) according to the manufacturer’s instructions.

Measurement of intracellular calcium [Ca2+

] i in PC12 cells:

Calcium plays important role in signal transduction as well as other cellular process such

as metabolic and physiological process. Even minute to minute changes in intracellular

calcium [Ca2+

] i levels can have a major concern on cellular activities. [Ca2+

] i was

measured by fluorometric analysis by using molecular probes fura-2 acetoxymetyl ester

pentapotassium (Fura2-AM; Sigma Aldrich, USA) .The PC12 cells were loaded with

3µmol/L fura2-AM in loading solution containing 125mM-NaCl, 5mM-KCl, 1.2mM-

MgSO4, 1.2mM-KH2PO4, 2mM-CaCl2, 6mM-Glucose, 25mM-Heps-NaOH(pH-7.4)

buffer at 37ºC &under 5% CO2 in incubator for 30 min. Unnecessary fura-2-AM was

removed by rinsing twice with titration solution- saline-A containing NaCl-8.182 g/L,

KCl-0.372 g/L, NaHCO3-0.336 g/L, Glucose- 0.9 g/L. The cells undergoing RV-10 µM,

NGF-50 ng/mL & combination of both (i.e. RV-10 µM + NGF-50ng/mL) or the control

group were represented in titration solution or saline A. [Ca2+

] i was measured by BMG-

fluoSTAR Omega at an emission wave length of 510nM by using a pair of excitation

were lengths at 355nM &380nM & (Fo) excitation was obtained at the ratio of that

generated by 355/380. Fmax was obtained after Cells were lysed with 0.1% Triton X-

100. Fmin was obtained after the addition of EGTA (5mM/liter final concentration). To

reduce the leakage of Fura-2AM, experimental setup were carried out at 35ºC. Ca2+

fluorescence were examined simultaneously at each time point (10 min, 20 min, 30 min,

40 min, 50 min and 60 min). All results shown are representative experiments from

three separate experiments under the same conditions and by the same procedure at each


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time point. The concentration of intra cellular calcium [Ca2+]i was calculated using the

following formula.

[Ca2+

]i = Kd X (Fo-Fmin) / (Fmax-Fo)

Where Kd is the dissociation constant of fura-2AM calcium complex and value is 224

nmol/L. The results were expressed in terms of percent of intra cellular calcium levels.

Western blotting experiments:

PC12 cells were seeded on poly-L-lysine-coated 75cm2 in normal serum medium for 24

h, then shifted to low serum (1% HS and 0.5% FBS) for 24 h and exposure to RV in

presence and absence of NGF for different time periods. Cells were washed with PBS,

scraped in ice cold PBS containing 1mM sodium orthovanadate,1% Sodium floride and

were pelleted, lysed using CelLyticTM

M Cell Lysis Reagent (Catalog no. C2978, Sigma,

USA) in the presence of protein inhibitor cocktail (Catalog no. P8340-5ML, Sigma,

USA), 1mM sodium-orthovanadate, 1mM DDT and incubated for 30 min at 40C. The

cell lysate was transferred to the micro centrifuge tube and centrifuged (15,000xg for 30

min) at 40C and the cell lysate was carefully transferred to the micro centrifuge tube.

The protein concentration was measured by the Bradford method (Thermo Scientific,

USA) using bovine serum albumin as a standard. Cell lysate (40 µg) was separated on

10% Tricine-SDS gel and transferred onto PVDF membrane (Millipore, USA) at 100V

for 2h at 40C. The membranes were blocked at 4

0C in TBST blocking buffer (5% non-fat

dried milk in TBS containing 0.1% Tween-20) for 2 h. Blots were incubated with the

appropriate antibodies overnight at 40C: anti-phospho-TrkA (1000), anti-TrkA (1000)

anti-phospho-ERK1/2 (1:1000),anti- ERK1/2 (1:1000), anti-phospho-p38 (1:1000), anti-

p38 (1:5000), anti-phospho-JNK (1:1000), anti-phospho-MEK1/2 (1:5000), anti-Rap1


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(1:1000),anti-phospho-Raf1 (1:1000),anti-phospho-BRaf (1:1000), anti-phospho-PKA

(1000), anti-phospho-CREB (1000) (Millipore, USA), anti-adnylate cyclase1 (1:500)

(Abcam, UK) and anti-β-actin (1:5000) (Sigma, USA). After three washes with TBST,

the membranes were then re-incubated for 2h at room temperature with secondary anti-

primary immunoglobulin G (IgG)-conjugated with horseradish peroxidase (Calbiochem,

USA). The blots were developed using luminol (Catalog No. 34080, Thermo Scientific,

USA) and densitometry for protein specific bands was done in Gel Documentation

System (Alpha Innotech, USA) with the help of Alpha EaseTM

FC Stand Alone V. 4.0.0

software. β-Actin was used as internal control to normalize the data. Exposures induced

alterations are expressed in relative term fold change in expression by comparing the

data with respective unexposed controls.

Morphological studies of RV induced neurite outgrowth in presence and absence

NGF:

Morphological analysis and quantification of neurite bearing were carried out using

phase-contrast microscope. Briefly, PC12 cells (1X105) were seeded on poly-L-lysine-

coated 6-well plates in the normal serum medium for 24 h. The medium containing low

serum (1% HS and 0.5% FBS) was replaced prior to exposure to RV, NGF and

combination of both for 4 days. One identical set was also run in presence of ERR1/2,

p38, cAMP antagonist and intracellular calcium chelator. After incubation, neuronal

differentiation of PC12 cell was observed under an inverted microscope using phase-

contrast objectives and photographed by the digital camera. At least 100 cells in each of

ten randomly separated fields were scored and the proportion of cells with neurites

greater than or equal to the length of one cell body were scored positive for neurite


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outgrowth, and expressed as a percentage of the total cell number in ten fields. The

neurite extension length was also measured for all identified positive neurite-bearing

cells in a field by tracing the longest length of neurite per cell using Image software

(Leica Q win).The value of neurite length (average maximal neurite length per neurite-

bearing cell in ten fields) was calculated and data from the ten fields in each well was

designated as one experiment. Experiments were repeated at least three times on

separate days and data are expressed as mean ±SD.

Statistical analysis:

Results were expressed as mean ± standard error of mean (SEM) for the values obtained

from at least three independent experiments. Statistical analysis was performed using

one-way analysis of variance (ANOVA) and Dunnett’s Multiple Comparison test using

Graph Pad prism (Version 5.0) software.

.


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Results

Biological interaction analysis of RV with membrane receptor:

It is known that NGF acts on tyrosine kinase receptor A (TrkA) and regulate neurite

outgrowth through activation of Ras-Raf-MEK1/2, which activates ERK1/2

(Gundimeda, et al., 2010., Haramoto, et al., 2008). To evaluate whether RV serves as a

small molecular agonist for TrkA, PC12 cells were treated with 10µM RV or 50ng/ml

NGF (as a positive control). As shown in figure-1a, significantly TrkA was activated by

NGF - but not in case of RV. In order to evaluate the biological interaction of RV with

membrane receptor was analyzed through in-silico experiment. Interestingly the binding

mode of RV followed the similar trend as with the FK (as well known activator of

adenylate cyclase) (figure-1b) suggesting that RV potentiated action of NGF may be

mediated through the adenylate cyclase activation. These findings corroborated with our

western blotting results where adenylate cyclase-1 was activated by 10 µM RV or 25

µM foskolin (figure-1c).

RV mediated activation of ERK1/2 and p38 phosphorylation:

MAPK pathways regulate cellular processes such as proliferation, survival/apoptosis,

differentiation, development, adherence, motility, metabolism, and gene regulation.

MAPK signaling modules have been identified in mammalian cells, with the three major

ones the ERK, the JNK, and the p38 MAPK pathways. To assess the activation of

ERK1/2, p38 and JNK1/2 MAP kinases in PC12 cells induced by RV, PC12 cells were

exposed with RV at various concentrations (5-100 µM) and NGF (50 ng/mL), as a

positive control for 1h. ERK1/2 and p38 pathways were activated at all the


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concentrations of RV while JNK pathway was not activated (figure-2a). When, the PC12

cells were exposed with 10 µM dose of RV at various time periods (15 min - 6 h), RV

induced the significant expression of phosphorylated ERK1/2 as early as 15 minutes of

exposure, attained peak by 30 min and sustained up to 2 h and gradually decreased from

4 h to 6 h. Expression of phosphorylated p38 was also increased within 15 min, and

gradually decreased from 30 min to 6 h. RV exposure could not modulate the expression

of phosphorylated JNK (figure-2b). We further investigated the activation of MAP

kinases by RV (10 µM) in presence and absence of NGF (50 ng/mL). As shown in

figure-2c, RV significantly increases the phosphorylation of ERK1/2, and p38. The

levels of phosphorylation of ERK1/2, and p38 were increased in combined exposures.

RV induced upregulated intracellular calcium levels [Ca2+

] i:

Calcium signaling is involved in many different intracellular and extracellular processes

ranging from synaptic activity to cell-cell communication and adhesion (Marambaud et

al., 2009). In the brain, calcium is essential for the control of synaptic activity and

memory formation, a process that leads to the activation of specific calcium dependent

signal transduction pathways and implicates key protein effectors, such as CaMKs,

adenylate cyclases, cyclic-AMP, MAPKs and CREB. (Greer PL, et al., 2008). To

determine the activation of Intracellular calcium levels [Ca2+

]i, cells were treated with

RV in the presence and absence of NGF. In RV exposure groups, after 10 min of

exposure the value was 500±9.52 and significantly increased from 530±10.15 to

610±9.04 after 20 - 50 min. A minor decrease was also observed for [Ca2+

] i level after

60 min of RV exposure, i.e. 550±16.8. In case of NGF, no significant changes were

observed at all the time periods but in co-exposure groups, the prominent changes in


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intracellular calcium were found ranging from 580±16.24 to 710±15.84 and it was

maximum at 50 min (figure-3).

RV promotes the cyclic-AMP activity:

Cyclic AMP, a secondary messenger, modulates cell growth and differentiation in

organisms from bacteria to higher eukaryotes (Tresguerres et al., 2011). To determine

the cyclic-AMP activity, PC12 cells were treated with RV or/and NGF. After 5min, the

value was recorded in control cells i.e. 10.65 pmol/mg protein and exposure of cells to

RV, NGF and combination of both significantly attenuated the levels of cAMP i.e.

21.72, 16.19 and 27.6 9pmol/mg protein respectively. The maximal attenuated levels of

cAMP were recorded at 30 min, the reading was 36.21, 21.72 and 44.74 pmol / mg

protein respectively. In addition, additive effect was found in co- exposure at all the time

periods (figure-4).

RV elevates Rap1, B-Raf, PKA and MEK1/2 phosphorylation without activation of

Ras:

The activation of various up-stream signaling molecules of ERK1/2 & p38 MAPKs

including Ras, Rap1,B-Raf, protein kinase A (PKA) and MEK1/2 were assessed

following the exposure of RV in presence and absence of NGF through western blotting

analysis. These molecules play pivotal role in proliferation, differentiation, synaptic

plastic and memory (Soren et al., 1998). RV activated Rap, B-Raf, PKA and MEK1/2

phosphorylation without activation of Ras. In co-exposures synergistic effect was found

in Rap, B-Raf, PKA and MEK1/2 phosphorylation (figure -5).


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RV induces the transcriptional factor CREB phosphorylation:

Several reports suggested that phosphorylation of transcription factors played critical

role for neurogenesis and neuronal differentiation (Scholzke, et al., 2007). To

investigate whether RV can activate these transcriptional factor, PC12 cells were treated

with RV (10 µM) in presence and absence of NGF (50 ng/mL). As shown in figure-6,

PC12 cells exposed to RV show increased expression of CREB as compare to

unexposed control. In co-exposures group, the expressions of these transcriptional

factors were increased and synergistic effect was observed (figure-6).

RV induces neuronal differentiation in PC12, the essentiality of ERK1/2 and p38

MAPKs:

We studied the involvement of ERK1/2 and p38 MAPKs in neuronal differentiation in

PC12 cells induced by RV. Prior to investigate the effect of ERK1/2(PD98059) and p38

(SB203580) inhibitors in neurite outgrowth, we identified the inhibitory doses of

ERK1/2 & p38 inhibitors for inhibition of ERK1/2 & p38 MAPKs induced by RV in

PC12 cell. It was observed that 20 µM of PD95059 and 10 µM of SB203580 blocked the

activation of ERK1/2 & p38 MAP kinases respectively by RV (figure-7a). The

morphological analysis was also done to assess the effect of these inhibitor on neurite

outgrowth following the exposures of RV or/and NGF (figure-7b). The quantification by

counting the neurite bearing cells and neurite elongation measurement was also done

using Leica Q win software. Figure-7c shows that RV, NGF and RV+NGF induced

significant neurite outgrowth (25.38±1.63, 38.03±1.95, and 43.82 ±1.71 µM) in

significant number of cell population (24.10±3.05, 36.04±2.11 and 42.17±3.89) at day 4

respectively. However, the neurite growth and neurite bearing cell number was


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comparatively less than that induced by RV, NGF and RV+NGF in presence of ERK1/2

and p38 inhibitors. In ERK1/2 inhibitor groups, the neurite outgrowth and neurite

bearing cell number was found in RV, NGF and RV+NGF (16.49±1.47, 20.97±1.64 and

22.05±1.59 µM neurite outgrowth length) and (16.03±2.12, 18.16±3.07 and 20.04±2.19

neurite bearing cell number) respectively at day 4. While, in case of p38 inhibitor

groups, RV, NGF and RV+NGF showed 19.47±1.39, 24.8±1.01 and 29.34±1.53 µM of

neurite outgrowth length and 19.1±2.01, 24.05±2.14 and 31.03±3.09 of neurite bearing

cell number respectively at day 4. However, inhibitory potencies of these two inhibitors

especially SB203580, on RV or/ and NGF induced neurite outgrowth are weaker than of

PD98059.

RV mediated induction in ERK1/2 and p38 phosphorylation requires intracellular

calcium [Ca2]i & cAMP:

To determine the role of intracellular calcium [Ca2]i & cAMP for activation of ERK1/2

and p38 phosphorylation. First, we identified the optimum concentration of BAPTA &

cAMPS-Rp for inhibition of intracellular calcium [Ca2] i & cAMP induced by RV in

PC12 cells and found 10 µM dose of BAPTA & 15 µM dose of cAMPS-Rp block the

activation of intracellular calcium [Ca2]i & cAMP respectively induced by RV (figure-

8a). After identification of inhibitory concentration, we treated cells with RV in presence

and absence of specific antagonists. As shown in figure-8b, exposure of cells to BAPTA

& cAMPS-Rp significantly inhibit the phosphorylation of ERK and p38 MAPKs when

compared to RV treated group. However, in both antagonists, BAPTA showed better

inhibitory potency in comparison to cAMPS-Rp. Further, we also checked the

expression of upstream signals of ERK and p38 MAPKs such as RAP1, B-Raf and PKA


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in presence of these antagonists. Results demonstrated that BAPTA only inhibited Rap1

and B-Raf without inhibition of PKA. While, cAMPS-Rp showed partial inhibition of

Rap1 and B-Raf and its inhibitory effect was more severe for PKA. The above finding

explained that both secondary messengers are responsible for ERK and p38 MAPKs

activation. These findings also explain that [Ca2]i induction was Rap1 and B-Raf1

mediated. While induction in cAMP was found to be primarily mediated by the

activation of PKA, Rap1, and B-Raf in PC12 cells. We further investigated the effect of

these antagonists in neuronal differentiation in PC12 cells. Cells were treated with RV

or/and NGF in presence and absence of BAPTA & cAMPS-Rp for quantification of

neurite outgrowth by phase contrast microscopy (figure-8c & d). Figure-8d showed that

RV, NGF and RV+NGF induced significant neurite outgrowth (24.62±1.40, 34.53±1.71,

and 42.39 ±1.10 µM) in significant number of cell population (25.10±2.05,

33.09±2.11and 44.15±2.99) at day 4 respectively. However, the neurite growth and

neurite bearing cell number was comparatively less than that induced by RV, NGF and

RV+NGF in presence of BAPTA and cAMP-Rp inhibitors. In BAPTA groups, the

neurite outgrowth and neurite bearing cell number were found in RV, NGF and

RV+NGF (14.75±1.10, 22.88±1.08 and 29.60±1.41 µM neurite outgrowth length) and

(15±1.12, 20±0.98 and 30±2.03 neurite bearing cell number) respectively at day 4.

While, in case of cAMP-Rp groups, RV, NGF and RV+NGF showed 17.34±1.53,

26.2±1.27 and 35.94±1.79 µM of neurite outgrowth length and 18±2.01, 25±2.98 and

36±1.97 of neurite bearing cell number respectively at day 4. Among both inhibitors,

BAPTA was more potent to inhibit neurite outgrowth in all treatment group.


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RV induces ERK1/2 and p38 phosphorylation via MEK in RAP1dependent and

PKA independent manner:

The data supported that ERK1/2 and p38 phosphorylation regulation represents a cross

point for several signaling pathways and is known to regulate a variety of genes in

neuronal functions. To unravel the mystery, the possible involvement of PKA, Rap1,

MEK1/2 signaling in RV unregulated CREB via ERK1/2 and p38 phosphorylation was

investigated by utilizing their molecular inhibitors. PC12 cells were treated with kinase

inhibitors including MEK1/2 Inhibitor U0126 (10 µM), PKA inhibitor H89 (15 µM),

Rap1 inhibitor GGTI 298 (5 µM) for 30 min then re-incubated with 10µM RV or alone

for 30 min and checked the expression of ERK1/2 and p38 phosphorylation and its up

and down stream signals such as Rap1, B-Raf, PKA MEK1/2 and CREB by western

blotting. We observed from the results that Rap1 inhibitor significantly block the

activation of Rap1, B-raf without affecting PKA activation, while PKA inhibitor

significantly induced the levels of Rap1 and B-Raf (Figure-9a). In both Rap1 and PKA

inhibitors, Rap1 inhibitor showed inhibition of ERK1/2 and p38 phosphorylation in

comparison to RV treated group. While PKA inhibitor failed to inhibit the activation of

ERK1/2 and p38 phosphorylation along with its upstream signals of B-Raf, MEK1/2.

(Figure-9a). The finding demonstrated that Rap1 is essential for ERK1/2 and p38

phosphorylation in PC12 cells in case of RV exposure. The results also demonstrate that

Rap1 mediated ERK1/2 and p38 phosphorylation crosstalk at MEK1/2. We further

proved this finding using MEK1/2 inhibitor (UO126) to check the expression of up and

downstream signals of MEK1/2. It was observed that MEK1/2 inhibitor significantly


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blocked the activation of MEK1/2 and its downstream molecules ERK1/2, p38 and

CREB without affecting its upstream signals (PKA, Rap1 and B-Raf) (figure-9b).


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Discussion

RV induces neurite outgrowth and potentiates the action of NGF to induce neurite

outgrowth in PC12 cells; however, the underlying mechanism is poorly understood. In

the present investigations, we show that (1). RV induces neurite outgrowth and

potentiates NGF-induced neurite outgrowth in PC12 through the AC-1 but not by TrkA.

(2). RV dose not potentiate NGF-induced activation of Ras and JNK; however (3).

Simultaneous treatment with RV and NGF acts in synergy to induce activation of

ERK1/2 and p38 and it’s up (cAMP, intracellular calcium, Rap1, B-Raf, PKA, MEK1/2)

and downstream molecules (CREB). These results indicate that RV-induced neurite

outgrowth involves neuritogenic action via activation of AC-1 and subsequent neurite

extension by ERK1/2 and p38 activation, and that although RV and NGF share common

intracellular events leading to neurite outgrowth, synergism between them arise, at least

in part, via activation of ERK1/2 and p38. This study is important because, in

neurodegenerative diseases, low concentrations NGF may be activating some pathways

but not all of those needed for neurogenesis. RV might activate these missing pathways

and thereby complement the action of NGF so that NGF even at a low concentration can

induce neurogenesis. This is particularly important, because, at sites of neuronal injury,

NGF is suboptimal for the induction of neurogenesis. Delivery of optimal concentrations

of NGF just to sites of neuronal injury is a daunting task (Gundimeda et al., 2010). Thus,

the NGF-potentiating activity of RV may be a highly useful tool in the treatment of

neuronal injuries.


in PC12 cells 2014

73

In PC12 cells, neurite outgrowth can be stimulated by diverse neurotrophic factors

activating corresponding receptors: Upon binding of NGF on Trk receptors, they

dimerizes and autophosphorylates, which results in triggering MAPKs (Scarpi et al.,

2012). However, prolong receptor-mediated activation of MAPK alone is insufficient to

induce neurite outgrowth. Activation of cAMP/PKA, intracellular calcium, and Rap1 are

also required for NGF-induced neurite outgrowth in PC12 cells. Intracellular cAMP is

enzymatically produced from ATP by adenylyl cyclases, either transmembrane adenylyl

cyclase (tmAC) or soluble adenylyl cyclase (sAC) (Kamenetsky et al., 2006), when,

NGF binds to TrkA, which activates sAC to produce cAMP (Stessin et al., 2006).

Subsequently, activated PKA and Epac converge to activate Rap1 (Stessin et al., 2006),

the aforementioned mediator of sustained MAPK activity (Yao et al., 1998 and York et

al., 1998). In contrast, FK binds tmAC to produce cAMP and activates PKA and B-

Raf/Rap1 in PC12 cells. While FK stimulation depends on the activation of the

MEK/ERK signaling but are PKA independent in PC12 cells. Our study In-silico

revealed that RV serves as a small molecular agonist for AC1 receptor and its binding

mode followed the similar trend as with the FK (figure-1b) suggesting that RV

potentiated the action of NGF mediated through the AC-1 activation. These data also

suggusted that RV induced PC12 cell neurite which is not associated with TrKA.

Intracellular calcium and cAMP are pleiotropic cellular regulators and both exert

powerful, diverse effects on cytoskeletal dynamics, cell adhesion and cell migration

(Howe et al., 2011). In this work, we clearly detected RV treatment induced intracellular

calcium and cAMP and showed synergist effect with NGF in PC12 cells (figure-3 & 4)..

Addition of BAPTA, a intracellular chelator inhibitor; and cAMP-Rp, a cAMP inhibitor,


in PC12 cells 2014

74

significantly blocked the potentiation of RV induced neurite outgrowth. On the other

hand, in RV plus NGF group, the effect these inhibitor (BAPTA and cAMP-Rp) was

less in comparison to NFG or RV treated alone. Moreover, our findings also indicated

the close correlation between intracellular calcium/cAMP and neurite outgrowth induced

by RV in PC12 cells (figure-8c & d) .

The activation of various signaling pathways, including PKA, Rap1 and B-Raf have

been linked with the control of de novo protein synthesis in the context of LTP (long

term, synaptic plasticity and memory) and converge the signal to CREB (Vitolo et al.,

2002). In this study, we found that RV significantly induced PKA, Rap1, B-Raf, CREB.

Moreover, we also found that RV showed synergetic effect with NGF to induce PKA,

Rap1, B-Raf, CREB without activation of Ras and JNK (figure-5 & 6).

It has been shown that through prolong activation of the MAPKs pathways, NGF

induces neurite outgrowth in PC12 cells. In the present study, RV can induce ERK1/2

and p38 activation for at least 6h at concentration as 10 µM and can act synergistically

with NGF to induce ERK1/2 and P38 activation (figure-2a, b & c). Furthermore, neurite

outgrowth induced by RV or/and NGF was inhibited by PD98059 and SB203580. These

results suggest that ERK1/2 and p38 are main mediater for neurite outgrowth in PC12

cells induced by RV or/and NGF. However, PD98059 was found more severe to inhibit

the neurite outgrowth in compare to SB203580 (figure-7b & c). These results suggust

that RV modulates MAPKs signaling pathways governing cell differentiation, in part

through activation of MAPKs.

As we know, FK induces MEK/ERK and P38 activation are Rap1 dependent and PKA

independent in PC12 cells. In present study, we found that like FK, RV induced


in PC12 cells 2014

75

MEK/ERK and P38 activation via Rap1 dependent and PKA independent in PC12 cells.

We also found that while cAMP or intracellular calcium inhibition, RV showed partial

inhibition of Rap1. Accordingly, these results showed that cAMP and intracellular

calcium, both provoked Rap1 activation in PC12 cells (figure-8b). On the other hand,

PKA inhibition (H89) enhanced RV-triggered Rap1 activation in PC12 cells (figure-9a).

In this study, we also found that whereas PC12 cells treated with various antagonists

such as, Calcium cheletor (BAPTA), cAMP inhibitor (cAMP-Rp) and Rap1 Inhibitor

(GGTI 298), only partly reversed RV-induced ERK1/2 and p38 activation, while PKA

Inhibitor (H89) was failed to inhibited ERK1/2 and p38 activation (figure-8b & 9a). This

finding indicates [Ca2]i, cAMP, Rap1 pathway might be involved in the RV-mediated

ERK1/2 and p38 activation. Moreover, our data also showed that RV could activate

ERK and p38 which could be abolished by U0126, a MEK1/2 phosphorylation inhibitor.

Additionally, RV- induced CREB activation was significantly affected by U0126

(figure-9b). Therefore, there is crosstalk between ERK, p38 MAPKs activation and RV-

induced intracellular calcium, cAMP/PKA/Rap1 pathway, CREB activation. Taken

together, these findings suggest that [Ca2]i/cAMP /Rap1- but not TrkA- or ERK, p38-

dependent, signaling pathway is involved in the mechanisms of RV-induced neurite

outgrowth.

Thus, RV promoted neurite outgrowth which was aroused via [Ca2]i and cAMP release

through activation of AC-1 and trigger the Rap1/ERK1/2/CREB) and p38 pathways.

Thus, the therapeutic strategy to stimulate neuronal cells events including proliferation,

migration and differentiation and neurite outgrowth are needed for several


in PC12 cells 2014

76

neurodegenerative disorders. Small molecules, such as RV may work as therapeutic

agents that possess the high neurotrophic potency.

RV Forskolin

AC receptor

Figure-1a. Interaction of RV with Trk-A. PC12 cells were seeded on poly-L-

lysine-coated 6-well plates in normal medium for 24 h and then shifted to low

serum medium (1% HS and 0.5% FBS) for 24 h prior to exposure to RV (10 µM) or

NGF (50ng/mL) for 5min and check the expression of p-TrkA and Trka by western

blotting as described in Materials and Methods.

Figure-1c. Interaction of RV with Adenylate cyclase1 Receptor. PC12 cells were

seeded on poly-L-lysine-coated 6-well plates in normal medium for 24 h and then

shifted to low serum medium (1% HS and 0.5% FBS) for 24 h prior to exposure to

FK (25 µM), RV (10 µM) or NGF (50ng/mL) for 5min and check the expression of

Adenylate cyclase1 by western blotting. β-actin was used as internal control to

normalize the data.

.

Figure-1b. Interaction of RV with Adenylate cyclase Receptor through in-silico

experiment. The docking studies were performed using Glide 5.6. The ligand RV

was prepared by LigPep 2.3 application using OPLS 2005 force field. The adenyle

cyclase protein (PDB Id: 1WWW) was prepared by protein preparation wizard

available in Schrodinger software package. The co-crystallized ligand (forskolin)

was selected for generation of docking grid in Glide5.6docking software and RV

was docked into the prepared grid (active site) using the automated ‘Extra

precision" (XP) mode of Glide5.6 to ensure the correct binding mode of the RV.

C FK-25µM RV-10µM NGF-50ng

Adenylate cyclase1

β-actin

C RV-10µM NGF-50ng/mL

p-Trk-A

Trk-A

p-ERK1/2

ERK1/2

p-p38

C 5μM 10μM 25μM 50μM 100μM 50ng/ml

RV-1h NGF-1h

p38

p-JNK

β-actin

p-ERK1/2

p-p38

p-JNK

C 15min 30min 1h 2h 4h 6h

ERK1/2

RV-10µM

p38

β-actin

Figure-2. Resveratrol activated ERK1/2, p38 phosphorylation concentration

and time dependent. a. PC12 cells were seeded on poly-L-lysine-coated 25 cm2

flask in normal medium for 24 h and then shifted to low serum medium (1% HS

and 0.5% FBS) for 24 h prior to exposure to RV different concentration

(5,10.25,50,100 µM) or NGF (50 ng/mL) for 1h and check the expression of

MAPKs by western blotting. β-actin was used as internal control to normalize the

data. b. PC12 cells were seeded on poly-L-lysine-coated 25 cm2 flask in normal

medium for 24 h and then shifted to low serum medium (1% HS and 0.5% FBS) for

24 h prior to exposure to RV (10 µM) for different time periods (30 min - 6 h) and

check the expression of MAPKs by western blotting. β-actin was used as internal

control to normalize the data.

Figure-2c. Resveratrol increased the phosphorylation ERK1/2 and p38 in

presence of NGF. PC12 cells were seeded on poly-L-lysine-coated 25 cm2 flask in

normal medium for 24 h and then shifted to low serum medium (1% HS and 0.5%

FBS) for 24 h prior to exposure to RV (10 µM) or/ and NGF (50 ng/mL) for 30 min

and check the expression of MAPKs by western blotting. β-actin was used as

internal control to normalize the data.

ERK1/2 and p38 MAPKs phosphorylation by Resveratrol

(a). (b).

C RV NGF RV+NGF C RV NGF RV+NGF C RV NGF RV+NGF

p-ERK1/2 p-p38 p-JNK

ERK1/2p38 β-actin

Figure-3. Flourometric analysis of Intracellular calcium levels in PC 12 cells

induced by RV in presence and absence of NGF. Intracellular calcium was

measured in PC 12 cells using Fura-2AM dye. Experimental method was described

in Materials and Methods. Values are mean ± SEM of three experiments each

carried out in triplicate. The values obtained were compared as indicated in the

figure by Dunnett’s test (*P < 0.05, **P < 0.01).

Figure-4. Resveratrol promotes the cyclic-AMP activity in PC 12 cells. PC12

cells were seeded on poly-L-lysine-coated 6-well plates in normal medium for 24 h.

The cells were then shifted to low serum (1% HS and 0.5% FBS) for 24 h and

exposure to RV (10µM) in presence and absence of NGF (50ng/mL) for different

time periods (5 min to 1 h). Experimental method was described in Materials and

Methods. Values are mean ± SEM of three experiments each carried out in

triplicate. The values obtained were compared as indicated in the figure by

Dunnett’s test (*P < 0.05, **P < 0.01).

Time Periods

0

5

10

15

20

25

30

35

40

45

50

5min 15min 30min 1h

Control

RV-10μM

NGF-50nM/mL

RV-10μM+NGF-

50nM/mL

**

**

*

*

**

*

**

**

pm

ol/

mg

pro

tein

Intr

acell

ula

r C

alc

ium

lev

els

(%

)

0

100

200

300

400

500

600

700

800

RV NGF RV+NGF

10min 20min 30min 40min 50min 60min

****

**** **

****

****

****

**

* *

Figure-5. Resveratrol activates Rap1, B-Raf, PKA and MEK1/2

phosphorylation without activation of Ras. PC12 cells were seeded on poly-L-

lysine-coated 25 cm2 flask in normal medium for 24 h and then shifted to low serum

medium (1% HS and 0.5% FBS) for 24 h prior to exposure to RV (10 µM) or/ and

NGF (50 ng/mL) for 30 min and check the expression of Rap1, B-Raf, PKA

,MEK1/2, Ras by western blotting. β-actin was used as internal control to normalize

the data.

Figure-6. Resveratrol induces the transcriptional factor expression CREB.

PC12 cells were seeded on poly-L-lysine-coated 25 cm2 flask in normal medium for

24 h and then shifted to low serum medium (1% HS and 0.5% FBS) for 24 h prior

to exposure to RV (10 µM) or/ and NGF (50 ng/mL) for 30 min and check the

expression of CREB by western blotting. β-actin was used as internal control to

normalize the data.

Figure-7a. Effect ERK1/2 inhibitor (PD98059) or p38 inhibitor (SB203580) for

activation of ERK1/2 or p38 respectively induced by RV. Confluent PC 12 cells

were serum starved for 6 hr and pretreated with PD98059 (20 µM) or SB203580

(10 µM) for 30 min and further exposed to RV (10 µM) for 15 to 6 h. In one set,

cells were also treated with NGF (50 ng/mL) for 15 to 30 min used as positive

control. ERK or p38 activation was determined by analyzing phosphorylated

ERK1/2 or p38 by Western immunoblotting.

p-p38

15min 30 min

RV 10 µM + SB203580 10 µM

p38

NGF 50 ng/mL

C 15min 30 min 1h 2h 4h 6

ERK1/2

p-ERK1/2

15min 30 min

RV 10 µM + PD98059 20 µM NGF 50 ng/mL

C 15min 30 min 1h 2h 4h 6

C RV NGF RV+NGF

Rap1

β-actin

β-actin

p-Braf

β-actin

p-MEK1/2

β-actin

Ras

β-actin

p-PKA

C RV NGF RV+NGF

C RV NGF RV+NGF

C RV NGF RV+NGF

C RV NGF RV+NGF

C RV NGF RV+NGFp-CREB

β-actin

Figure-7.(c). Neurite Bearing and elongation measurement in PC12 cells at day

4 induced by RV or/and NGF in presence and absence of ERK1/2 inhibitor

(PD98059) or p38 inhibitor (SB203580). Neurite outgrowth was determined as

indicated in materials and methods. The results are shown as the mean ± SE for

three experiments. The values obtained were compared as indicated in the figure by

Dunnett’s Multiple Comparison test (*P < 0.05, **P < 0.01).

Neu

rite

elo

ng

atio

n (

µM

)

Neu

rite

bea

rin

g c

ells

05

101520253035404550 Normal

PD98059

SB203580

*

***

*

****

**

05

101520253035404550 Normal

PD98059

SB203580

***

*

*

Figure-7.(b). Morphological analysis of neuronal differentiation of PC12 cells

at day 4 induced by RV or/and NGF in presence and absence of ERK1/2

inhibitor (PD98059) or p38 inhibitor (SB203580). Cells were grown in poly-L-

lysine-coated 6 well plate and then shifted to low serum medium (1% HS and 0.5%

FBS) for 24 h, and pretreated with PD98059 (20 µM) or SB203580 (10 µM) for 1

hr then further incubated with RV (10 µM) and/or NGF (50 ng/mL).

CONTROL NGFRV RV+NGF

CONTROL+PD NGF+PDRV+PD RV+NGF+PD

CONTROL+SB NGF+SBRV+SB RV+NGF+SB

Figure-8. (b). Effect Calcium cheletor (BAPTA) or cAMP inhibitor (cAMP-Rp)

for activation PKA, Rap1, B-Raf, p38 and ERK1/2 induced by RV. Confluent

PC 12 cells were serum starved for 24 hr and pretreated with BAPTA (10 µM) or

cAMP-Rp (15 µM) for 30 min and further exposed to RV (10 µM) for 30 min and

check the expression of PKA, Rap1, B-Raf, p38 and ERK1/2 by western blotting.

β-actin was used as internal control to normalize the data.

Figure-8. (a). Effect Calcium cheletor (BAPTA) or cAMP inhibitor (cAMP-Rp)

for activation of intracellular calcium or cAMP respectively induced by RV.

Confluent PC 12 cells were serum starved for 24 hr and pretreated with BAPTA (10

µM) or cAMP-Rp (15 µM) for 30 min and further exposed to RV (10 µM) for 5 min.

Intracellular calcium or cAMP activation was determined as indicated in materials

and methods. The values obtained were compared as indicated in the figure by

Dunnett’s test (*P < 0.05, **P < 0.01).

0

100

200

300

400

500

600

Control Control+BAPTA RV RV+BAPTAIntr

a C

ellu

lar

Ca

lciu

m le

vels

(%

)

**

0

5

10

15

20

25

Control Control+cAMP-Rp RV RV+cAMP-Rp

**

*

pm

ol/

mg p

rote

in

C RV RV

BAPTA-10µM

+C RV RV

cAMP-Rp-15µM

+

p-PKA

Rap1

p-Braf

p-p38

β-actin

p-PKA

Rap1

p-Braf

p-p38

β-actin

p-ERK1/2 p-ERK1/2

Figure-8c. Morphological analysis of neuronal differentiation of PC12 cells at

day 4 induced by RV or/and NGF in presence and absence of intracellular

cheletar (BAPTA) or cAMP inhibitor (cAMP-Rp). Cells were grown in poly-L-

lysine-coated 6 well plate and then shifted to low serum medium (1% HS and 0.5%

FBS) for 24 h, and pretreated with BAPTA (10 µM) or cAMP (15 µM) for 1 hr then

further incubated with RV (10 µM) and/or NGF (50 ng/mL).

Figure-8d. Neurite Bearing and elongation measurement in PC12 cells at day 4

induced by RV or/and NGF in presence and absence of intracellular cheletar

(BAPTA) or cAMP inhibitor (cAMP-Rp). Neurite outgrowth was determined as

indicated in materials and methods. The results are shown as the mean ± SE for

three experiments. The values obtained were compared as indicated in the figure by

Dunnett’s Multiple Comparison test (*P < 0.05, **P < 0.01).

Neu

rite

elon

gat

ion

(µ

M)

Neu

rite

bea

ring

cel

ls

05

101520253035404550 Normal

BAPTA

cAMP-Rp

*

*****

*

*

05

101520253035404550 Normal

BAPTAcAMP-Rp

***

***

*

*

CONTROL NGFRV RV+NGF

CONTROL+BAPTA NGF+BAPTARV+BAPTA RV+NGF+BAPTA

CONTROL+cAMP-Rp NGF+cAMP-RpRV+cAMP-Rp RV+NGF+ cAMP-Rp

Figure-9b. Effect MEK1/2 inhibitor (UO126) for activation of ERK1/2, p38

and its up and downstream signaling molecules induced by RV. Confluent PC

12 cells were serum starved for 24 hr and pretreated with UO126 (10 µM) for 30

min and further exposed to RV (10 µM) for 30 min and check the expression of

PKA, Rap1, B-Raf, p38, ERK1/2, MEK1/2 and CREB by western blotting. β-actin

was used as internal control to normalize the data.

Figure-9a. Effect Rap1 Inhibitor (GGTI 298) or PKA inhibitor (H89) for

activation of PKA, Rap1, B-Raf, p38 and ERK1/2 induced by RV. Confluent

PC 12 cells were serum starved for 24 hr and pretreated with GGTI 298 (5 µM) or

H89 (15 µM) for 30 min and further exposed to RV (10 µM) for 30 min and check

the expression of PKA, Rap1, B-Raf, p38 and ERK1/2 by western blotting. β-actin

was used as internal control to normalize the data.

p-PKA

Rap1

p-Braf

p-p38

β-actin

C RV RV

U0126-10µM

+

p-MEK1/2

p-CREB

p-ERK1/2

p-PKA

Rap1

p-Braf

p38

β-actin

C RV RV

H89-15µM

+

p-PKA

Rap1

p-Braf

p-p38

β-actin

C RV RV

GGTI-5µM

+

p-ERK1/2 p-ERK1/2

Figure10. Signaling mechanism by which RV synergies the activity of NGF to

induce neuronal differentiation in PC 12 cells. The RAS⁄RAF⁄MEK⁄ERK

pathway is activated by Nerve growth factor (NGF) binding to receptor tyrosine

kinase (RTK), which leads to the activation of the small G-protein RAS.

Subsequently, RAF, MEK and ERK are activated in a cascade of phosphorylation

events. Through the phosphorylation of many targets, MAPKs regulate cell fate.

Resvertrol (RV) lead to the activation of adenylyl cyclase and incres the levels of

intracellular calcium, activated adenylyl cyclase converts ATP into cAMP. The

second messenger cAMP acts through many effectors and has many cellular effects.

While intracellular calcium increase the levels the small G-protein, Rap1 via

Rap1/B-Raf signaling complex. These two pathways interconnect or crosstalk at

MEK1/2. This allows cells to regulate the distribution, duration, intensity and

specificity of the response.

Trk

ARaf1

MEK1/2

ERK1/2

MAPKKKK

MAPKKK

MAPKK

MAPK

Nerve Growth Factor

P

P

P

Ras

Jnk1/2Pp38P

Gene Transcription

Neuronal Differentiation

B-Raf

PKA

P

P

AC

1

cAMP

RV

[Ca]i

Rap1P

CREB

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