Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Both, the cytokine Tumor Necrosis Factor-α (TNF-α) and the enzyme cytosolic
phospholipase A2 (cPLA2) are crucial driving forces in mediating the cellular
inflammatory response and are involved in ischaemic injury. During an ischaemic
insult, TNF-α is endogenously generated. Apart from the recognized effects of TNF-
α, such as the induction of apoptosis, proliferation and differentiation, if present in
low dosages, it also mediates cytoprotective effects. Upon activation, cPLA2
contributes to the ischaemic challenge with the generation of mediators of cellular
injury and apoptosis. Upon stimulation, this calcium dependent enzyme translocates
to the phospholipid compartment of the cell membrane and induces the hydrolysis of
sn-2 ester bonds in phospholipids. It governs the release of free fatty acids and
lysophospholipids and generates role players of inflammation. We suggest a role for
cPLA2 in the TNF-α mediated cytoprotection, with a distinct phosphorylation and
translocation pattern.
Aims
The involvement of cPLA2 in TNF-α mediated cytoprotection in the C2C12 murine
skeletal muscle cell line in tolerance to ischaemia was examined. To investigate the
nature of the cPLA2 phosphorylation pattern, the mitogen activated protein kinases
(MAPKs) p38 and extracellular regulated kinase (ERK) as contributors to cPLA2
phosphorylation and activation, were examined at appropriate time points. To dissect
out the cPLA2 interplay and dependencies with these MAPKs within the pathway
context, the selective cPLA2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3)
was employed and its effect on cell viability was examined. Fluorescence microscopy
was used to substantiate cPLA2 activation, by assessing its cellular distribution,
translocation and cell organelle target preference, using co-localization and z-stack
techniques. In addition, the induction of the apoptotic pathway through analysis of
caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage was examined. The
role of caspase-3 in cPLA2 turnover was addressed employing the caspase inhibitor,
Z-DEVD-FMK. Methods
Cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) with 10% fetal
bovine serum (FBS), and incubated under 5% CO2 conditions, until 50%-70%
confluent. Using DMEM supplemented with 1% horse serum, cell differentiation into
myotubes was induced. Differentiated cells were preconditioned for 30 min
classically, with 0.5 ng/ml TNF-α or the cPLA2 selective inhibitor AACOCF3 (10
μM) respectively. Followed by a 60 min washout period the cells were subjected to 8
hrs simulated ischaemia. Cellular viability; and cPLA2 phosphorylation- and
translocation events were assessed using Western blots and advanced
immunocytochemistry and imaging techniques.
Results
Preconditioning with TNF-α, ischaemic preconditioning; and the use of the cPLA2
inhibitor AACOCF3, attenuated the decrease is cell viability brough about by
ischaemia. Western blot analysis indicates the induction of the apoptotic pathway with
caspase-3 and PARP cleavage. A significantly reduced translocation of pcPLA2 to the
nuclear region in the TNF-α preconditioned group compared to the ischaemic group,
as reflected by reduced mean nuclear fluorescence intensity, was observed. A z-stack
analysis confirmed that the nuclear and endonuclear region was the target organelle
for cPLA2. 3-dimensional co-localazation analysis of pcPLA2 with the nuclear marker
nucleoporin p62 mirrored these results.
Discussion and conclusion
Our results provide evidence that there is a role for cPLA2 in TNF-α mediated
cytoprotection. Although we do not observe a differential activation pattern in terms
of cPLA2 phosphorylation at various time points within the ischaemic event, and no
differential inactivation of cPLA2 via caspase-mediated cPLA2 cleavage, we describe
a differential cPLA2 translocation pattern, similar to that in IPC. Through inhibition of
cPLA2 translocation, a functional cPLA2 inhibition might be achieved. This would
imply inhibition of the inflammatory pathway and a subsequent reduction in the
generation of inflammatory mediators. In addition we describe an effect of TNF-α
preconditioning on the efficacy of the caspase inhibitor Z-DEVD-FMK. Our results shed light on the survival mechanisms employed by the ischaemically challenged cell
in a setting of TNF-α mediated cytoprotection. This might lead to novel approaches in
the context of inflammation treatment, through agents that control differential cPLA2
trafficking within the cell. / AFRIKAANSE OPSOMMING: Beide, die sitokien “Tumor Necrosis Factor-α (TNF-α)” en die ensiem, sitosoliese
fosfolipase A2 (cPLA2) is uiters belangrike bemiddelaars van die sellulêre
inflammatoriese respons en is verder ook betrokke by isgemiese selskade. TNF-α
word endogeen gegenereer tydens ‘n isgemiese intervensie. Afgesien van ‘n
verskeidenheid effekte, soos die inisiëring van apoptose, sel-proliferasie en -
differensiasie, bemiddel dit ook selbeskermende meganismes indien dit in lae
konsentrasies in die sel teenwoordig is. Na aktivering dra cPLA2 by tot die isgemiese
intervensie deur die vorming van bemiddelaars van selskade en apoptose. Hierdie
kalsium-afhanklike ensiem translokeer na die fosfolipied membraankomponent na
stimulering en induseer die hidrolise van die sn-2 esterbinding in die fosfolipied. Die
vrystelling van vry vetsure en lisofosfolipiede word sodoende bewerkstellig wat
verder gemetaboliseer kan word tot inflammatoriese bemiddelaars. Ons stel voor dat
cPLA2 ‘n rol in TNF-α bemiddelde selbeskerming speel en dat dit gepaardgaan met
kenmerkende fosforilerings- en translokeringspatrone.
Doelwitte
Die rol van cPLA2 tydens TNF-α bemiddelde selbeskerming is in ‘n C2C12
skeletspiersellyn na blootstelling aan isgemie ondersoek. Die rol van die MAPKs, p38
en ERK, is ondersoek om vas te stel of hulle betrokke is by die aktivering van cPLA2.
Die selektiewe cPLA2 inhibitor, AACOCF3, is gebruik om te bepaal of die
fosforilering van MAPKs ook cPLA2-afhanklik is. Die sellulêre cPLA2 verspreiding,
translokering en teiken selorganelle is ook ondersoek met behulp van fluoresensie
mikroskopie deur gebruik te maak van ko-lokalisering en z-plaat tegnieke. Verder, is
die indusering van die apoptotiese paaie ondersoek deur tegnieke wat kaspase- en
PARP kliewing meet. Die kaspase inhibitor, Z-DEVD-FMK, is gebruik om vas te stel
of kaspase-3 ‘n rol speel in cPLA2 kliewing in ons selmodel.
Metodes
Selle is gekweek in Dulbecco’s gemodifiseerde Eagles Medium (DMEM) waarby
10% fetale kalf serum (FBS) gevoeg is, en wat geïnkubeer is in 5% CO2 totdat dit
50%-70% konfluent was. Die selle is verder gedifferensieer in miobuise deur gebruik te maak van DMEM waarby 1% perdeserum gevoeg is. Gedifferensieerde selle is vir
30 min klassiek geprekondisioneer asook respektiewelik met 0.5 ng/ml TNF-α en die
cPLA2 selektiewe inhibitor, AACOCF3 (10 μM). Na ‘n 60 minute uitwas periode is
die selle blootgestel aan 8 h gesimuleerde isgemie. Sellulêre lewensvatbaarheid,
cPLA2 fosforilering- and translokering is ondersoek deur onderskeidelik gebruik te
maak van die “Western” klad metode en gesofistikeerde immunositochemiese beeld
tegnieke.
Resultate
Prekondisionering met TNF-α, isgemiese prekondisionering asook inhibisie van as
cPLA2 met die inhibitor, AACOCF3, het ‘n beduidende toename in
sellewensvatbaarheid tot gevolg gehad. Daar is ook dmv die “Western” klad tegniek
bewys dat apoptose geïduseer word deur middel van kaspase-3- en PARP kliewing.
Daar is insiggewend minder translokasie van cPLA2 na die nukluêre fraksie in die
isgemiese groep in vergelyking met die TNF-α geprekondisioneerde groep
waargeneem (die gemiddelde nukluêre fluoreserende intensiteit is bepaal om
voorafgaande feit te staaf). Die cPLA2 teiken organel is geverifieer as die nukleus en
die endonukluêre gebied deur middel van z-plaat analises. Drie-dimensionele kolokaliserings
analises van pcPLA2 met die nukluêre merker, nucleoporin p62 het
hierdie resultate bevestig.
Bespreking en Gevolgtrekking
Ons resultate verskaf bewyse vir ‘n rol vir cPLA2 in TNF-α bemiddelde
selbeskerming. Alhoewel daar nie ‘n differensiële aktiveringspatroon in terme van
cPLA2 fosforilering tydens verskeie tydspunte in die isgemiese intervensie
waargeneem is nie, en ook geen kaspase-3 bemiddelde kliewing van cPLA2 nie, word
‘n differensiële translokeringspatroon soorgelyk aan die isgemiese
prekondisioneringsgroep, waargeneem. Funsksionele cPLA2 inhibisie kan dus
moontlik bewerkstellig word deur inhibisie van cPLA2 translokasie. Die
inflammatoriese respons kan dus moontlik so inhibieer word en die vorming van
minder inflammatoriese bemiddelaars tot gevolg hê. Verder het TNF-α
prekondisionering ook ‘n effek op die effektiwiteit van die kaspase-inhibitor, ZDEVD-
FMK. Ons resultate werp ook lig op die meganismes wat deur selle onder isgemiese toestande uitgeoefen word tydens TNF-α bemiddelde selbeskerming.
Hierdie resultate mag lei tot nuwe benaderings in die konteks van behandeling teen
inflammasie deur gebruik te maak van middels wat cPLA2 translokering in die sel
beheer.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/17377 |
| Date | January 1900 |
| Creators | Loos, Benjamin |
| Contributors | Engelbrecht, Anna-Mart, Smith, Rob, University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xxv, 118 leaves : ill. |
| Rights | University of Stellenbosch |
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