Calcium Signaling and the Expression of Nuclear Genes
Encoding Mitochondnal Proteins
Martino Di Carlo
Thesis submitted to the Faculty of Graduate Studies in partial fulfilrnent of the
requirements for the degree of
Master of Science
Graduate Programme in Kinesiology and Health Science
York University
Toronto, Ontario, Canada
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Calcium Signaling and t h e E x p r e s s i o n
o f N u c l e a r Genes E n c o d i n g M i t o c h o n d r i a l
Proteins
by
M a r t i n o D i Car10
a thesis submitted to the Faculty of Graduate Studies of York
University in partial fulfillment of the requirements for the degree
of
Master o f S c i e n c e
1998 6
Permission has been granted Io the LIBRARY OF YORK
UNIVERSITY to lend or seIl copies of this thesis, to the
NATIONAL LJBRARY OF CANADA to microfilm this thesis and to
lend or seIl copies of the film, and to UNIVERSITY
MICROFILMS to publish an abstract of this thesis.
The author reserves other publication rights, and neither the
thesis nor extensive extracts from it may be printed or otherwise
reproduced without the author's written permission.
Abstract
We have previously demonstrated that cytochrorne c transcription is upregdated by
an A23 187-induced increase in cytosolic Ca" concentration. The increased expression of
cytochrome c appears to involve a protein kinase C (PKC)pathway. To evaluate which PKC
isoform is involved, we co-transfected L6E9 muscle cells with the -326 cytochrome c
promoter-CAT constnift as well as calcium-sensitive(a and P,J or insensitive (6 and 5) PKC
isoform expression vectors. The results demonstrated a 1.5- ,2.1-, and 2.0-fold enhancement
of the A23 187 effect for PKCa, PKCP,,, and PKC C, respectively. To determine if mitogen-
activated protein kinase (MAPK) was acting downstream of PKC, we examined the time
course of MAPK activation in response to A23 187 treatment by using a phospho-specific
MAPK antibody. MAPK activation increased after 1 hou, was maximal at 2 hours and
declined to approximately 50% of maximum by 4 hours. Pre-treatment of the cells with
PD98059,a MEK inhibitor, attenuated the A23 187 effect on cytochrome c tramactivation
by 37i15% (n=5). To determine the connection between PKC and MAPK, cells were COtransfected
with expression vectors encoding the Ca" -sensitive isoform, PKCa, a
dominant-negative Raf mutant (RaBOl)or wtRaf, dong with the -326 cytochrome c
promoter-CAT constnict. The results showed that PKCa acts through Raf to activate MAPK,
since Raf30 1 reduced A23 187-mediatedtranscription of cytochrome c by 3% 10% ( ~ 4 - 6 ) .
niese data suggest that Caz+-mediatedincreases in cytochrorne c trans-activation in muscle
ceus are due, in part, to the activation of a PKC-MAPK pathway. Since the ~a"-res~onsive
region of the cytochrorne c promoter possesses binding sites for Spl and Zi868,we
iv
examined their roles in cytochrome c transcriptional activation in response to A23 187.
ûverexpression of Sp 1 repressed cytochrome c transcriptional activation in vehicle treated
cells by 4417% (n=7), but this was partially reversed by A23187 treatment. Forced
expression of Zif268 enhanced cytochrome c transcription by 2.7-fold, in a Ca"-dependent,
but DNA binding independent manner (n=5). Thus, Ca" activates signaling pathways,
possibly including PKC-MAPK, leading to the activation of Zif268 and enhanced
transcription of cytochrome c. These data help us to understand a portion of the intracellular
signaling pathways involved in t n g g e ~ gmitochondrial biogenesis in muscle cells.
Ac knowledgements
I would like to thank my s u p e ~ s o rDr.
, David Hood, for his guidance and support. I wouid
also like to thank Dr. Damien Freyssenet for allowing me to contribute to and continue the
work he began on calcium signaling, while a pst-doctoral fellow in Dr. Hood's laboratory.
I am also grateful to the other members of Dr. Hood's lab and to my fkiends in the biology
department, pariicularly John Andreucci, Mark Gagliardi and Natalie Rodrigues.
1 am especially grateful to my farnily for their support during al1 my years as a university
student. I dedicate this work to the memory of my father, Antonino Di Car10 (December 4.
1932- March 12, 1997).
Table of Contents
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vi
Tableofcontents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vii
ListofFigures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ix
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .x.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.
Review of Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Mechanisms of Ca" Release and Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.
A . Plasma Membrane Calcium Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
A .1 Voltage-operated calcium channels (VOCs) . . . . . . . . . . . . . . . . . . . . . . . . . .6
A.2 Receptor-operated calcium channels (ROCS) . . . . . . . . . . . . . . . . . . . . . . . . . .7
A.3 Store-operated calcium channels (SOCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
B . intracellular Calcium Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
B.1 Ryanodine Receptors (RyR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
B.2 Lnositol Triphosphate Receptors (IP,R) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
C. Calcium Uptake ChannelsPumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.
C.1 ~ a ' l ~ aexchanger
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C.2 Sarco(endo)plasmic reticulum Ca2'-~TPasePumps (SERCA) . . . . . . . . . . 10
C.3 Plasma membrane C ~ " - A T P ~Purnp
S ~ (PMCA) . . . . . . . . . . . . . . . . . . . . . 10
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D.Mitochondna
E . Calcium Ionophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I I
F.Sumrnary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I l
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Cytochrome c Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
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A . Cytochrome c Promoter
13
B . Cytochrome c expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
B.1 Electrical Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
B.2 CAMP-mediatedCytochrorne c Transcription . . . . . . . . . . . . . . . . . . . . . . .15
B.3Thyroid Hormone and Cytochrome c Expression . . . . . . . . . . . . . . . . . . . . . 15
Calcium and Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
A . Calciun Responsive Elements .......................................... 16
A .1 CAMPResponsive Elements (CRE) ............................... 16
A.2 S e m Responsive Element ( S E ) ................................ 16
B . Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
SignalTransduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
A-ProteinKinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
A .1 Protein Kinase C (PKC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
vii
A.2 Mitogen-Activated Protein Kinases (MAPKs) . . . . . . . . . . . . . . . . .2 1
A.3 Proline-rich tyrosine kinase 2 (PYK.2) . . . . . . . . . . . . . . . . . . . . . . .22
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B. Protein Phosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
B .1 Mitogen-activated protein kinase phosphatase- 1 (MW- 1) . . . . . . . . 25
B.2 Calcineurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
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Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
CellCulture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
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A23187Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
Transient Transfection and Chlorarnphenicol Acety!transferase (CAT) Assay . 28
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Northem Blot Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
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Western Blot Analyses
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Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .-.3 0
...
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . ..
.. . . . . . . . . . . . . . . . . . . vil1
Results
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
PKC overexpression in L6E9 skeletal muscle cells. . . . . . . . . . . . . . . . . . . . . . . 31
Co-transfection of PKC and Raf expression vectors . . . . . . . . . . . . . . . . . . . . . .31
MEK is involved in A23 187-mediated increases in cytochrome c
transcriptional activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
ERK 1 and 2 are activated in response to A23 187 treatment . . . . . . . . . . . . . . . .34
Overexpression of Sp 1 inhibits transcriptional activation from
pRC3CATl-326 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
Overexpression of Zif268Egr- 1 increases transcriptional activation
from pRC4CATI-326 in a calcium-dependent manner, independent
ofDNAbinding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
Zif 268 mRNA, but not protein, is induced by A23 187 treatrnent . . . . . . . . . . . .37
A23 187 treatment reduces the steady-state levels of glutamate
dehydrogenase (GDH) mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
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FutureWork . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
Appendix A - Raw Data and Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Appendix B - Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5 7
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
viii
List of Figures
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.
Overview of Experirnental Mode1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Review of Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.
Calcium Transport in a Skeletal Muscle Ce11 . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Partial Map of the Rat Cytochrome c Promoter . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Schematic Representations of PKC Isoforms . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Schematic Diagram of a Protein Kinase Cascade . . . . . . . . . . . . . . . . . . . . . . . . 24
Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
The effect of overexpressing PKC isofoms on cytochrorne c
transcriptional activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32
Co-transfection of PKCa and Raf on cytochrome c transcriptional
activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
Co-transfection of PKCC and Raf on cytochrome c transcriptional
activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
The role of MEK in A23 187-mediated cytochrome c tramactivation . . . . . . . . . 35
Time course of MAPK activation in response to A23 187 . . . . . . . . . . . . . . . . . .35
Overexpression of Sp 1 inhibits transcriptional activation
ofcytochromec . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36
Overexpression of Ep-1 on cytochrome c transcriptional activation . . . . . . . . . 38
The effect of A23 187 on Zif268 mRNA levels . . . . . . . . . . . . . . . . . . . . . . . . . .39
The effect of A23 187 on Zif268 protein levels . . . . . . . . . . . . . . . . . . . . . . . . . .40
The effect of DMSO on GDH mRNA levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
The effect of A23 187 on GDH mRNA levels . . . . . . . . . . . . . . . . . . . . . . . . . . .42
The full length rat cytochrome c prornoter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
List of Abbreviations
A23 187
ATF-2
CAT
CF
CRE
CREB
CSK
DAG
DNA
Egr- 1
EGTA
ERK
ERiSR
FAK
FM06
FKBP 12
GDH
p,
P,R
M-1
LPA
MAPK
MEK
mRNA
N'FAT
NFKB
NIH 3T3
NRF- 1
p70d"
PD98059
PIPZ
PKA
PKC (c, n, or a)
PLC
PM ATPase
P W
ROCC
RYR
SERCA
Antibiotic 23 187 (calcium ionophore)
Activation of Transcription Factor-:!
Chloramphenicol Acetyltransferase
Calcium Influx Factor
CAMPResponsive Element
CRE Binding protein
c-Src Kinase
Diacylglycerol
DeoxyribonucIeic acid
Early Growth Response factor- 1
Ethylene Glycol-bis-Tetraacetic Acid
Extracellular signal Regulated Kinase
Sarco(endo)plasmic Reticulum
Focal Adhesion Kinase
Calcineurin lnhibitor
FK506 binding protein (1 2 lcilodaltons)
Glutamate Dehydrogenase
inositol trisphosphate
hositol Trisphosphate Receptor
Jun N-terminal Kinase- 1
Lysophosphatidic Acid
Mitogen Activated Protein Kinase
Mapk/Erk Kinase
messenger ribonucleic acid
Nuclear Factor of Activated T-cells
Nuclear Factor kappa of B-lymphocyte
Mouse-derived fibroblast ce11 line
Nuclear Respiratory Factor- 1
Ribosomal Protein S6 Kinase
Synthetic MEK Miibitor
Phosphatidylinositol4,5-bisphosphate
Protein kinase A
Protein Kinase C (conventional, novel, or atypical)
Phospholipase C
Plasma Membrane C a 2 ' - ~ ~ P a spump
e
Proline-rich tyrosine kinase-2
Receptor Operated Calcium Channels
Ryanodine Receptor
Sarco(endo)plasmic Ca2'-~TPasepump
SOCC
SOS
SPI
S E
S rc
~ 6 2 ~ ' ~
TPA
VOCC
Zia68
Store Operated Calcium Channels
Son of Sevenless
Simian V i n s Protein- I
S e m Responsive Element
Sarcoma of Rous Chicken
Temary Complex Factor
Tetradecanoyl-Phorbol- 13-Acetate
Voltage Operated Calcium Channels
Zinc Finger clone #268
Introduction
The plasticity of muscle has k e n studied ushg whole anUnal and ce11 culture models
for many years. One aspect of this is how skeletal and cardiac muscle adapt to increases in
functional demand imposed by stimuli, such as endurance training. Perhaps the most
important alteration in muscle phenotype which under lies increased Functional capacity in
response to endurance training is rnitochondrial biogenesis.
Mitochondnal biogenesis is a complex process involving the synthesis of
phospholipids, transcription and translation of nuclear- and mitochondrially-encoded genes,
and protein import (45). Many investigators have employed rnodels designed to mimic
endurance training to demonstrate that the functional adaptation is mediated by
mitochondrial biogenesis (45, and references therein). A question that remains to be
answered is: What is the signal or signals that triggers mitochondrial biogenesis in response
to chronic muscle use? This is obviously too complex to study in its entirety, therefore the
focus of this thesis is on the transcriptional regulation of mitochondrial biogenesis.
Specifically, an important question which will be addressed is: what cellular event during
chronic contractile activity could regulate the transcription of nuclear genes encoding
proteins destined for import into mitochondria? One possible candidate is the movement of
Ca" ions.The cyclic movement of Ca2+ions into and out of the cytosol is a critical event in
the contraction of all types of muscle. An overview of how calcium was manipulated in our
mode1 is shown on Page 5.
Calcium is a second intssenger that mediates a diverse array of cellular processes
( 1 1). One of these is transcriptional regulation. Both the localization of the Ca" increase (43)
and the amplitude and duration of calcium signals (28) c m differentially activate gene
expression. in order to regulate transcription, cytosolic calcium signals m u t be propagated
to the nucleus. One potential factor involved in such a pathway is protein kinase C (PKC).
The PKC family consists of 11 isoymes which have been ciassified into conventional,
novei, or amical subgroups. These distinctions are based on primary structure and CO-factor
requirements (73). The conventional PKCs (a,P,, fi,, y) require Ca" and diacylglycerol
(DAG) for activation (73). Therefore, conventional PKCs (cPKCs) are the most likely
candidates involved in ~a"-mediated signalling. However. this does not exclude the
possibility that Ca" may indirectly activate other PKC isozymes via $Ta
activated
protease, such as calpain (3 1, 53, 92).
Our previous work has s h o w that Ca" signals up-regdate the transcription of
cytochrorne c (37) and repress the expression of glutamate dehydrogenase (GDH) . The
increased transcription of cytochrorne c appears to be mediated via a PKC-dependent
pathway since the effect of Ca" is abolished by prolonged pretreatrnent with 11-0-
tetradecanoyl-phorbol-13-acetate(TPA) (37). However, the specific PKC isozyme involved
in the Ca2'-rnediated response is not known. Preliminary data have also demonstrated that
a mitogen activated protein kinase (MAPK) is involved downstream of PKC in the Ca2-mediated regulation of cytochrome c. A probable factor linking PKC to MAPK is Raf- 1,
since a direct connection between PKCa and Raf-1 has been established in MH3T3
fibroblasts (55).
The transcription factor which is responsible for the enhancement in the expression
of cytochrome c in response to increased intracellular Ca" concentration is unknown. In
order to address this question, the regions of DNA upstream of the cytochrome c gene must
be known. The cytochrome c promoter has been cloned and a series of constmcts with
various regions of the promoter fused to the bactenal chloramphenicol acetyltransferase
(CAT) gene were created (33). The Ca"-responsive element appears to be located in the
region fiom -66 to + 1 15 of the cytochrorne c promoter (37), which contains an Sp 1 binding
site (32). The ~a"-responsive element was identified through the use of deletion constmcts
of the cytochrome c promoter (37).
The Ca2'-responsive site lies somewhere in the region between -66 to 1 15 base pairs
of the promoter and fint intron (37). ï h i s region contains a consensus binding site for only
one transcription factor, Spl (32). This fact obviously suggests that Spl is a potential
candidate in the search for the transcription factor involved in the transcriptional regulation
of cytochrorne c. However. another possible candidate transcription factor is Zif268IEGR- 1.
Zia68 is an immediate early gene which encodes a transcription factor known to bind a GCrich element, very similar to the Sp 1 binding site (16). Recent work suggests that Sp 1 and
Zia68 can cornpete with each other for a binding site or bind cooperatively, depending on
the promoter context (46, 60). It has been suggested that Zif268 acts as a repressor of gene
expression (17)when it binds ta upstream promoter regions. interestingly, the analysis of
the promoter of another nuclear gene encoding a mitochrondrial protein, glutamate
dehydrogenase (GDH),reveals that it contahs a Zif268 binding site (23). Since it is also
known that Zif268 is responsive to elevations in intraceilular calcium (1), an evaluation of
3
the response of GDH to A23 187 treatment will provide a valuable contrast to the behaviour
of cytochrome c, as well as provide an index of the time course of the response.
Based on this background, the specific purposes of this thesis are the following:
1.
To identi@ which specific PKC isoform is involved in cytochrome
c expression, and whether Raf-l and MAPK have roles in this
signaling pathway in skeletal muscle.
2.
To investigate the time course of the A231 87 effect on Zif268 and
GDH expression.
3.
To determine the effect of overexpression of Zif268 and Sp 1 on the
transcriptional activation of cytochrome c.
Overview of Experimental Mode1
m yoblasts
myotubes
transfect
differentiate
A23187
--)
Ca2+
Sarcolemma
t
[Ca 2+]
Review of Literature
Mechanisms of Ca2+Release and Uptake
Calcium is one of the most highiy regulated ions in living cells. Resting fiee Ca2+is
approximately 100 nM,while extracellular Ca" concentrations can be as high as 2 mM (10,
18). The necessity for controlling Ca" levels within cells is critical due to the role of Ca"
in many cellular processes, such as ce11 growth and differentiation, muscle contractility and
cell death ( 10. 18,66). This section will provide a brief overview of the mechanisms through
which cells handle CaT' ions.
A. Plasma Membrane Calcium Channels
-4./ t hirage-operarrd crrlciitni chcrnnels (VOCCs)
VOCCs are mainly located in excitable cells such as muscle and nerve cells (10).
VOCCs are comprised of a number of distinct calcium channels which are activated by
depolarization of the plasma membrane. Al1 the VOCCs have voltage sensing regions which
respond to changes in membrane potential by switching to a channel-open conformation. ( 18)
Individual VOCCs are classified according to their e!ectrophysiological and
pharmacological properties. T-(tiny) type channels are activated and inactivated at low
membrane potentials (44). ïhese channels play a role in initiating action potentials
@acemaking)(68). The remaining VOCCs, namely B-(brain) type, L-(long lasting) type, N-
(neither L nor T) type and P-(purkinje) type, are activated at higher membrane potentials (-30mV) and have a relatively long open-time compared to T-type channels (44. 68). The
high voltage channels were differentiated from each other based on their susceptibility to
various toxins and calcium channel blockers.
A.? Receptor-operared cdciitm chonnels (ROCCs)
ROCCs are activated by binding of agonists to various receptors on the plasma
membrane ( 10). The ROCCs c m be divided into hvo distinct classes. The first class consists
of charnels in which the receptor and the channel coexist in a single molecule or closely
associated n~oiecules.The second class consists of channels which are not in direct contact
with the receptors. Receptor activation generates second messengers which act on the
calcium channels (68).
A.3 Store-operoied cnlcizm chonnels (SOC'Cs)
SOCCs respond to the Ca" content of the ER'SR. Activation of SOCCs has been
described as 'capacitative entry', which refers to the influx of calcium fiom the extracellular
space in response to the emptying of the EWSR ( 1 1 ).
SOCCs exist in excitable and non-excitable cells. These charnels are encoded by
transient receptor potential (trp) and trp-like (trpl) genes in Drosophila ( 1 9). Homologous
genes have been identified in micr and humans (9). The exact mechanism by which these
calcium-release activated channels (CRAC) are activated is not presently known. Several
hypotheses are currently being considered ( 19): 1) direct coupling between the EWSR and
the SOCCs, 2) the involvement of GTP-binding proteins; 3) the production of a diffusable
second messenger known as the calcium influx factor (CF)in response to emptying the
ER'SR, 4) the activation of protein phosphatases, and 5) increased cGMP leading to the
activation of cGMP dependent protein kinases.
B. Intracellular Calcium Channels
B. 1 Rj*anodineReceptors (RyR)
Ryanodine receptors are calcium channels Iocated in the sarcoplasrnic reticulurn (SR)
of muscle cells and in the endoplasmic reticulurn of other cells. There are three ryanodine
receptor isoforms. The different isofonns were identified by the order in which they were
cloned. and their tissue distribution. RyR 1 (skeletal type), followed by RyR2 (cardiac type)
and RyR3 (brain type).
Ryanodine receptors are composed of four identical subunits (tetramer) arranged to
form a 'four-Ieaf clover' structure (36). There is high sequence homology (669'0) between
al1 three RyR isoforms with the carboxy terminus forming the transmembrane region and the
amino terminus forming the large cytoplasmic domain, known as the 'foot' structure (66).
'The RyR subunits are associated with FK506 binding protein 1%Da (FKBP 12) in a 1 :1 ratio.
FKBP 12, which is expressed at high levels in al1 types of muscle, is believed to act as a
stabilizing factor for the ryanodîne complex (36,66).
The activity of RyR channels is modified by a variety of agents (36). Micrornolar
calcium and millimolar adenine nucleotides act to open the RyR channel, resulting in a rapid
movement of calcium into the cytosol. Many pharmacological agents are also known to
modulate the RyR channel. Caffehe ai concentrations in the millimolar range activates
calcium release through RyR channels, while rutheniurn red can block calcium release &orn
the SR. Other agents, such as ryanodine and doxorubicin, have dual effects depending on
their concentrations (3 5).
B.2 hosifol Triphosphate Recepfors (IP,R)
IP, receptors are similar to RyRs in amino acid sequence and structure ( 1 1, 36).
Calcium release boom the SR through the P3Rsoccun by the binding of LP, to the LP3R. [P,
is generated by the hydrol~sisof PiP, by phospholipase C (PLC). PLC activation occurs
through the G-protein-linked receptors and tyrosine-kinase linked receptors ( 1 1, 66). P,R
have been identified in cardiac and skeletal muscle, however, IP,Rs do not play a major role
in E-C coupling (66).
C. Calcium Uptake Channels/Pumps
C. I . V i . TU-'
' exchanger
The ~ a ' X a ' +exchanger is a membrane protein which plays a role in calcium
homeostasis in excitable cells. The exchanger can function as a bi-directional electrogenic
punip. depending on the ion gradients and membrane potential (49). in the 'fonvard' mode.
the exchanger transports 3 Na* ions into the ce11 and rernoves 1 cal' ion. The exchanger cm
function in 'reverse' mode by transponing 1 ~ a into
" the ce11 and removing 3 Na' ions (7).
Na-/Ca2' exchanger a c t i v i ~was fust identified in squid axon and has been identified
in a number of different tissues such as brain, heart and muscle (49). It has been extensively
studied in vertebrate cardiac muscle. Both 'forward' and 'reverse' exchanger activity has
' ~ in
been observed (7, 61). This exchanger plays a critical role in controllhg ~ a levels
cardiac cells, which is an important determinant of contractility (74). The N ~ Y c ~ 'exchanger
'
has recently been identified in mouse skeletal muscle (7). No appreciable 'reverse' mode
transport activity was identified in mouse skeletal muscle fibres. However, 'fonvard' mode
exchanger activity does appear to be involved in Ca" handling in skeletal muscle (7).
C.7 Sarco(endo)plasmic reliclclum C d * - ~ T P a sPumps
e
(SERCA)
SERCA pumps belong to a family of C a " - ~ ~ ~ aenzymes
se
that catalyze the
translocation of Ca" f?om the cytoplasm into the lumen of the sarcoplasrnic reticulum (67).
This protein consists of a cytosolic domain, a 'stalk' region, and a transmembrane domain
(26). The SERCA pump altemates between 2 states in the process of pumping Ca" into the
lumen of the SR (64,65). in the E l state, the enzyme binds 2
ca2+ions and
1 ATP. The
subsequent hydrolysis of ATP causes a conformational switch to EZ and the release of the
2 ~ a ions
"
into the lumen of the SR (64).
C.3 Plast~iamembrane CU'* -A TPase Pump (PMCA)
The plasma membrane C a ' = ~ f ~ a spump
e
is present in most eukaryotic cells (87).
This protein is another component of the intncate system which keeps Ca'- concentrations
below 100 nM in resting cells (15). The PMCA is a member of a family of P-type ATPases,
which includes the SERCA pumps ( 1 5). The activity of this ~ a ' *pump is regulated by the
binding of caimodulin ( 15. 87). It binds ~ a and
" ATP in a 1:1 ratio and extrudes calcium out
of the ce11 upon ATP hydrolysis (1 5). The activity of the PMCA is modulated by protein
kinase A (PKA) and protein kinase C (PKC). Phosphorylation of the
ca2+
pump by these
kinases increases the maximum velocity of the C ~ " - A T P ~reaction
S~
(87).
resulted in increases in the steady-state mRNA levels of the proto-oncogenes c-fos and c-myc
in human peripheral blood lymphocytes (42). This demonstrated that ~ a "signals can
activate the expression of transcription factors, which can potentially alter the expression of
a nurnber of other genes leading to new phenotypes. Some evidence for the role of ca2+
in
phenotype transitions in skeletal muscle exists (56). These researchers treated primary
skeletal muscle cultures with A23 187, which resulted in a sustained 10-fold increase in
intracellular ~ a levels
"
maintained for 16 days. The A23 187 treatment induced a reversible
transition in the myosin profile of the muscle cells, changing fiom fast to slow myosin
isofonns (56).
F. Surnrnary
This section has provided a b i e f overview of the complex systern of channels and
organelles involved in Ca2* mobilization. Vanous stimuli. such as depolarization or the
binding of agonists. such as bradykinin or endothelin. can result in the mobilization of
calcium fiom both extracellular spaces andor the SRER. Elevations in cytosolic calcium
are usually transient. The activities of the
C ~ ' + - A T P ~ S ~and
S
the Na /C$ exchanger
remove calcium from the cytosol. Figure 1 is a schematic diagram of the components
involved in regulating calcium levels within cells.
Calcium Tlsaisport in a Skeletal Muscle CeU
Figure 1 : A schematic diagram of calcium transport channels in a ce11 adapted h m Bemdge. et al. ( IO).
Cytochrome c Gene Expression
A. Cytochrome c Promoter
The somatic cytochrome c gene encodes 3 mRNA transcripts of 1400,1100, and 700
nucleotides (33). Cytochrome c is known as a 'housekeeping' gene, however, recent work
suggests that cytochrome c expression is controlled at the level of transcription
(88, 89, 90). in order to investigate transcriptional control of cytochrome c , a detailed
analysis of the promoter rnust be available. The full length promoter was cloned and used to
identib several regulatory elemenü through deletion analysis (33). A schematic diagram of
a pan of the cytochrome c promoter is shown in Figure 2 .
.
l
1
CE-1
'
NRF-1
exon 1
CCAAT
cytochrome c
SPI
Pdapted from Evans, MJ. and RC.Scarpulla J. 6id.Chem 264:14361-14368, 1989
Figure 2: Partial map of the nt sornatic cytochrome c promoter
B. Cytochrome c expression
B. / Eiectrical S~irn~tlntion
Electrical stimulation models have k e n fiequent1y used to investigate the regulation
of cytochrome c expression in cardiac and skeletal muscle (88, 89,90). mRNA analysis and
reporter assays were used to demonstrate that electrical stimulation induces the transcription
of c-Fos. c-Jun and Nuclear Respiratory Factor4 (NRF-1)pnor to the induction of
cytochrome c expression (88). Subsequent experiments employed reporter assays and
electrophoretic mobility shiA assays (EMSA) to identifi NRF- 1 and c-Jun as key factors in
mediating cytochrome c transactivation in response to electrical stimulation ir. cardiac
myocytes (88). Perhaps the most interesting fmding was the involvement of c-Jun on
cytochrome c expression. since there are no consensus Jun binding sites on the cytochrorne
c promoter. Antibody 'supershi fis'confirmed that c-Jun binds to CAMPresponsive elements
(CRE) located on the cytochrome c promoter (88).
Electrical stimulation aiso affects the expression of cytochrome c through a posttranscriptional mechanism (90). Chronic stimulation of rat hindlimb muscle induced
increases in cytochrome c mRNA and protein (90). The chuiges in cytochrome c mRNA and
protein appeared to be the result of changes in mRNA stability and/or translation efficiency
(90). EMSA with a riboprobe representing part of the 3' untranslated region (UTR)of
cytochrome c was used to identim changes in RNA-protein interactions. Electrical
stimulation resulted in decreased RNA-protein interaction via an as yet uncharacterized
'inducible inhibitory factor' (90).
B.2 CAMP-rnediatedCytochrorne c Transcription
The cytochrome c promoter contains two CRE cis-elements (4 1). Expenments were
conducted to determine whether these CRE sites can mediate the transcription of
cytochrome c. Balb/3T3 cells that were treated with agents that increase CAMP levels
resulted in increases in cytochrome c mRNA. A number of techniques were employed to
establish that the cytochrome c mRNA increases were due to transcriptional activation
caused by CRE-CREB interactions (4 1 ). However, CAMP-CREB mediated transcription
appears to be cell-type specific, since there was no effect of CAMP on cytochrome c
transcription in COS- 1 cells (32).
B.3 Thvroid Hormone and Cytochrorne c E-rpression
Thyroid status is an important regulator of oxidative metabolism due to its effect on
mitochondrial biogenesis (78). The administration of 3,5,3'-triiodo - L - thyronine (T,) to
Sprague-Dawley rats resulted in a delayed increase in cytochrome c mRNA (78). This change
in cytochrome c expression was attributed to increased transcription by the use of nucfear
transcription assays. However, alterations in mRNA stability were also apparent (78).The
mRNA levels remained elevated despite declines in transcription at approximately 24 hours
following T, admininstration (78). Thyroid status is also an important regulator of
C M O C ~ ~c O
expression
~ ~
during muscle development. Hypothyroidism affected both
transcriptional and post-transcriptional regulation of cytochrorne c expression in a tissue
specific manner (85). The hypothyroid condition resulted in decreases in steady-state mRNA
in heart without affecthg protein levels, whereas, in muscle there was no change in mRNA
but the protein content was reduced (85).
Calcium and Gene Expression
Calcium signals participate in a wide array of cellular processes, some of which are
mediated by the activation of transcription factors (1 1,28,40). The involvement of calcium
signals in so many processes brings forth the question of specificity. What are the
mechanisms by which cells 'interpet' calcium signals and initiate the signaling pathways
which result in the correct response? This question is partially answered by the identification
of calci um-responsive elements and calcium-activated transcription factors.
A. Calcium Responsive Elements
A. I CAMP Responsive EIemenrs ('CRERE)
Calcium entry into neurons induces the phosphorylation of the CRE binding protein
(CREB) on serine- 133. This phosphorylation event is associated with increased transcnption
of c-fos (10).n i e kinase that phosphorylates CREB is unknown, however, it is believed to
reside in the nucleus (40). Some potential candidates for the kinase that phosphorylates
CREB are: calmodulin-kinase IV. p70SbK, and protein kinase A (10). CRE-mediated
transcription of c-fos by CREB is triggered by elevations of nuclear calcium and is not
sensitive to changes in cytosolic calcium (43).
A. 2 Serum Responsive Elentent (SM)
The S M is the target of a group of proteins cornmonly referred to as the temary
complex. This complex consists of a serum response factor (SRF) dimer bound to a
monomer ( ~ 6 2 ~ of
' ~the
) ETS domain-containing family of transcription factors (40). The
identification of the SRE as a calcium-sensitive element was the result of studies on c-fos
16
transcription, however, the details of how calcium signals are transmitted to the S E are
unknown (40). One potential pathway involves the mitogen- activated protein kinase
(MAPK). Neurotransmitîer-mediated increases in cytosolic calcium has been implicated in
the activation of the Ras-MAPK pathway (6).The involvement of these cytosolic factors in
SRE-rnediated transcription is in agreement with the fmding that activation of transcription
through the SRE is triggered by elevated cytosolic calcium (43). MAPK activation is linked
to SRE-mediated transcription via Elk- 1 (40). Elk- 1 is a member of the ETS domain family
of transcription factors, which is phosphorylated by the MAPKs in response to growth factor
stimulation (9 1).
B. Transcription Factors
The question of the specificity of the response to calcium signals has been recently
addressed (78). A key aspect to understanding how this specificity is achieved is to consider
the nature of calcium signals. Calcium signals can Vary in parameters such as fiequency.
duration, and amplitude, depending on the stimulus and the extracellular calcium
concentration (1 1, 18, 28). ~ a ' -signals were manipulated in B-lymphocytes, generating
transient spikes or kept ~ a continuously
' ~
elevated, in order to examine the activation of
NFKB, JNK l / ATF-7, and NFAT (28). These transcription factors responded differently to
the transient calcium spikes. NFKB and JMWATF-2 remained active after the the ~ a "
s i p a l was teminated by the use of EGTA, whereas, NFAT required a continuous elevation
of Ca" to remain in the nucleus (28). There was also a difference in the sensitivity of these
transcription factors to ~ a signals.
"
A low, sustained elevation o f cytosolic Ca2*activated
17
N'FAT, but did not activate NFKBor JNK 1/ATF-2 (28).
Signal Transduction
A. Protein Kinases
A. 1 Protein Kinase C (PKC)
PKC comprises a farnily of 1 1 isozymes (72). The different isozymes are classified
into three groups based on pnmary sequence and regdation (69,72). The conventional PKCs
(cPKC) are comprised of PKC a.
P,, a,, and y. This group is CaL'- and phorbol
ester-
sensitive. The novel PKCs (nPKC) are activated by phorbol esters, but do not have Ca"
binding sites (72). The third class, atypical PKCs (aPKC) do not have phorbol ester or Ca2'
binding sites (69, 7 2 ) . The primary structures of the three classes of PKC are s h o w in
Figure 3.
PROTEIN KINASE C ISOFORMS
PHORBOL ESTER
PSEUDOSUBSTRATE
PHOSPHAT IDYLSERiNE
SUESTRATE
CALClUM
HINGE
ATYPICAL
Adaptcd from A.C.Neiwon,/. Biol. Chem. 270: 28495-28498, 1995.
Figure 3: Schrmatic representation of the PKC isofoms. The binding sites for phorbol esters, phospholipids.
ATP and substrates are indicated.
Regulation of conventionai and novel PKCs has been well characterized (72).
Activation of these classes of PKC invoive the removal of the pseudosubstrate region, which
masks the active site of these enzymes (72). Molecules such as diacylglycerol (DAG) or
phorbol esters c m initiate the removal of the pseudosubstrate and cause the translocation of
the PKC isozyrne to the plasma membrane (69,72). The PKC isozymes can then interact
with membrane phospholipids such as phosphatidy lserine or phosphatidylcholine for full
activation (72). Ca" ions function as CO-factorsserving to increase the aKmity of the cPKCs
for phosphatidylserine (72). Atypical PKCs require phospholipids such as phosphatidylserine
and phosphatidylinositol-3,4-bisphosphate for activation, however, how they are reguiated
in vivo remains to be determined (71,92).
PKC isozymes usually phosphorylate serine/threonine residues in basic sequences of
their substrates (72). There is a growing body of evidence that implicates PKC as an activator
of MAPK signaling pathways (8. 50, 55. 59, 79, 86). Raf-l was identified as a potential
substrate for PKC through the use of imrnunoprecipitation and in vitro kinase assays (55).
AI1 the cPKCs (a,P, y) were capable of phosphoryiating Raf- 1 in vitro (55). This group first
proposed the involvement of PKC in the mitogen-activated protein kinase pathway. The
proposed signaling cascade was as follows: PKCa-Raf- 1-MEK-MAPK-TCF (55).
The ability of members of cPKCs, nPKCs, and aPKCs to activate the ERK class of
MAPKs has been recently Livestigated (79). Lmmunoprecipitation and in vitro h a s e assays
were empioyed to demonstrate that al1 the PKCs (constitutively active mutants) tested, a, P,,
6, E, q, and C, could activate MEK and ERK2 MAPK (79). Further analysis revealed that
members of the cPKCs and nPKCs induce MEK activation via Raf-1. The atypical PKC
<
also activated MEK and MAPK, however PKC C activates MEK through a distinct pathway,
not involving Raf-l (79).
A. 2 Mirogen-A crivaf ed Protein Kinases (MAPh)
The mitogen-activated protein kinases (MAPKs) are a family of proteins that are
involved in relaying extracellular signals to the nucleus (l2,24) The three main classes of
MAPK are: extraceliular signal-regulated kinases ( E u s ) , jun N-terminal kinases
(Ms)/stress-activated protein kinases (SAPKs), and p38 MAPK. Al1 the MAPKs are
activated by phosphorylation at conserved threonine and tyrosine residues, however, certain
levels of specificity exist. Different stimuli activate particular classes of MAPK and the
active MAPKs have distinct targets (12). Although there is some specificity. the study of
MAPK signaling pathways is complicated by interactions between components of various
pathways, often referred to as 'cross-talk' (12).
The ERKs are perhaps the niost extensively studied of the MAPKs. ERK I (p44) and
ERK 2 (p42) are present in a wide variety of tissues and celi Iines (76). ERK activation is
associated with mitogen stimulation, however, they are activated by many non-mitogenic
stimuli (76). The upstream components involved in activating the ERKs involve the protein
kinases Ras, Raf and MEK (1 2,24). Once activated, the ERKs translocate to the nucleus and
phosphorylate specific transcription factors such as Elk-1 (24,76). Interestingly, active ERKs
can phosphorylate the protein kinases, Raf-1 and MEK (76). However, the significance of
this activity is not currently known.
A.3 Proline-rich iyrosine kinase 2 (PYK2)
PYK2 is a recently cloned non-receptor tyrosine kinase isolated fiom a human brain
complementary DYA library (57). PMU is a protein of approximately 1ZOkDa, structurally
related to Focal Adhesion Kinases (FAKs) (34, 57). Northem blot and in situ hybridization
analyses were employed to investigate the expression of PYK2 (57). P M U mRNA was
detected in brain and kidney. in situ hybridization revealed differential expression of PYK2
mRNA in rat brain structures (57). These investigators did not show or discuss data
conceming PYKZ expression in other tissues.
The investigation of PYKî continued, focussing on how this tyrosine kinase is
regulated. These researchers treated Pheochromocytoma cells (PC 121, a neuronal-derived
ce11 line, with agents such as carbachol and KCl and assayed PYK2 activation by
imrnunoprecipitation and imrnunoblotting with an anti-phosphotyrosine antibody. Both
treatments induced phosphorylation of P Y W in a calcium dependent manner (57). The role
of calcium was established by the use of the calcium chelator, EGTA, and the calcium
ionophore A13 187 (57). Additional work revealed that PYK2 activation was also linked to
phorbol-ester treatment and G-protein coupled receptor stimulation by agonists such as
bradykinin and lysophosphatidic acid (LPA) (25,27, 57).
The next logical step regarding P W was to investigate what lies downstream of
it. The work of the Schlessinger laboratory provided some insights into a connection between
PYK2 and the ERK MAPKs (27, 57). The picture that has emerged, mostly fiom
immunoprecipitation and immunoblot experiments, is that PYKî foms complexes with the
22
adaptor proteins GrbZ and Shc, which then recruit the guanine nucleotide exchange factor
SOS, leading to the activation of Ras and ultimately to the MAPKs (27, 57). An important
factor in PYK2-induced MAPK activation is the non-receptor tyrosine kinase Src. Active
PYK2 autophosphorylates on tyrosine-402, which provides a docking site for Src, and
possibly activates it (27). Although the furiction of Src in this context is not yet clear, it does
appear to play a role in LPA- and bradykinin-induced activation of MAPK. Over-expression
of Csk. a protein tyrosine kinase that inhibits Src activation, reduced MAPK activation in
response to those treatments (27). Figure 4 is a schematic diayam of the involvement of
calcium in regulating vanous protein kinases, such as PYK2 and PKC,and also shows the
interactions between some of the signaling components.
Figure 4: A schematic diagram demonstrating protein kinases uansmining a sipal âom outside the ceil to rhc
nucleus. resulting in gene espression.
B. Protein Phosphatases
B. 1 iMirogen-activatedprotein kinase phosphatase- 1 (Mm-1)
An important aspect of signal transduction involves the role of protein phosphatases.
These phosphatases c m inactivate protein kinases by removing phosphate groups fiom
serine/threonine or tyrosine residues (82). A family of phosphatases has recently been
identified which specifically dephosphorylates the MAPKs (8 1). One member of this family
is the immediate early gene, MKP- 1, which may be a physiologicai regulator of the ERK
class of MAPK (20, 8 1).
The potential role of MKP- 1 in regulating signaling through the ERK MAPKs has
generated a great deal of interest, since both ERK activation and MW-1 expression are
induced by growth factor stimulation (20, 8 1, 83). However, recent work has demonstrated
that MKP-1 expression was the result of growth factor-induced increases in cytosolic
calcium (8 1). It seems plausible that the ERKs might play a role in MKP- 1 expression, since
Ca2+selectively activates the ERKs (81), however, this does not appear to be the case.
Inhibition of ERK activation with PD98059 did not repress MKP-I expression (8 1). The
pathway linking Ca" signals to MKP-1 expression is not currently known.
B. 2 Calcineurivl
Calcineurin is a Ca2'-calmodulin dependent protein phosphatase expressed in a wide
variety of tissues. This enzyme is a heterodirner composed of catalytic and calmodulin
binding subunits, coupled to a Ca2'-binding regulatory subunit (54), which is inhibited by
the immunosuppressive agents cyclosporin A and FK506 (54, 62). Activated calcineurin
dephosphorylates the transcription factor, NF-AT, which causes NF-AT to translocate to the
nucleus and activate transcription of immune response genes in T- and B-lymphocytes (62,
83).
Recent work has demonstrated a role for calcineurin-NF-AT signaling in cardiac and
skeletal muscle (1 6. 70). Ca2*signals had previously been implicated as a potential factor in
cardiac hypertrophy (70. and references therein). The involvement of calcineurin and NF-AT
was convincingly demonstrated through the generation of transgenic mice expressing
constitutively active forms of these factors (70). M A T , in combination with the cardiac
specific factor GATA. activated cardiac gene expression in the transgenic mice. The hearts
of these mice were approximately 2-fold larger than the control mice, a phenotype which was
completely reversible by treating the transgenic mice with cyclosporin A (70).
Calcineurin also appears to regulate transcription in skeletal muscle (16). Fibre-type
transformation has been of interest to muscle physiologists for many years. Recent work has
demonstmted that Ca2+can induce revenible fibre-type transitions in primary skeletal muscle
cultures (56). The molecular b a i s of these transitions involves signaling through calcineurin
and NFAT activation. Experimental evidence demonstrates that M A T acts in concert with
26
myocyte enhancer factor-:! (MEFî) to selectively activate the expression of genes which
confer a slow phenotype on skeletal muscle ( 16). Further support of this mode1 was provided
by the observation that inhibition of calcineurin by cyclosporin A induces slow to fast
transitions ( 16).
MATEFUALS AND METHODS
troductiorl
This section contains a description of the experimental techniques employed. A detailed
outline of the methods is available in Appendix B.
l2cuauc
L6E9 myoblasts were cultured on 1OOmm plastic dishes in DMEM containing 10%
Fetal Bovines Serurn (FBS) and 1 % penicillin/streptomycin (PB) at 37°C in a 5% CO,
inciibator. At approxirnately 60-70% c o ~ u e n c edifferentiation
,
was induced by switching
to Dulbecco's Modified Eagle's Medium containing 5% horse senun (heat-inactivated). The
cells were left in differentiation media for 6-8 days. Fresh media was added every 48 hours.
A33 187 Treatmerg
A23187 (Sigma) was added to the cultures following differentiation (>7S0h
myotubes) at a final concentration of 0.75pM for the indicated tirne points. Al1 experiments
conducted include vehicle-treated @MSO) controls.
henicol Acetyltrmferase K A T ) A s ç a ~
L6E9 myoblasts were transfected once the cells reached approximately 60%
confluence (29, 77). CMV-Pgal was co-transfected with the DNA constmct of interest to
control for differences in transfection efficiency. AAer A23 187 treatment (48 hours),
myotubes were rinsed with PBS and scraped. Cell extracts were prepared as described
previousiy (37). The CAT reactions were nomalized to P-galactosidase activity and
incubated for 2-2.5 hours. Acetylated chloramphenicol was resolved by thin layer
28
chromatography and the percent conversions of the acetylated forms were detemhed by
electronic autoradiography.
Total RNA was isolated fiom L6E9 myonibes by the guanidium thiocyanate method
(38). Equal arnounts of total RNA were electrophoresed on 1% agarose, 5% formaldehyde
gels and transferred to nylon membranes (Hybond N, Arneaham) by capillary blotting. BIots
were prehybridized for 2 h o m and hybridized for 16-24 hours at 42 OC. cDNA probes were
radiolabelled with a"~-dCTP (Amesham) and a random primer labelling kit W B ) .
Washing conditions for each specific probe are indicated. Al1 blots were probed with 18s
rRNA to correct for loading differences. Blots were exposed to film at -70°C for
autoradiography.
Western Biot Analyses
At the appropnate tirne following A23 187 treatment, myotubes were rinsed with cold
PBS and scraped in 2 0 0 ~ of
1 2x Laemmli Buffer (62.5m.M Tris-HCl, pH 6.8, 20% glycerol,
2% SDS, 5% P-mercaptoethanol) containhg protease inhibitors. Extracts were sonicated
(3x 10 second pulses), heat denatured at 95 O
C
for 5 minutes and spun briefly to pellet
insoluble material. Protein concentrations were determined (13). Equal arnounts of whole
ce11 extract were electrophoresed on 10% polyacrylarnide-SDS gels and transferred to nylon
membranes by electrotransfer. Membranes were blocked for 1 hour in 5% milk. Primary
incubations were carried out at 4°C ovemight. Signals were detected by the use of the
appropnate secondary antibody coupled to horseradish peroxidase (HRP)and enhanced
29
chemilluminescence according to manufacturer's instructions (Amersham).
Data were analyzed using unpaired Student's t-tests or the appropriate ANOVA
design. Unequal groups in factonal designs were dealt with by canying out both Type iIi and
Type I analyses.If the following conditions are met. then the Type iIi analysis is valid: 1) the
F ratios are identical, 2) the p-levels are below 0.05. and 3) data loss is moderate (35). Tukey
HSD for unequal N was used to identify significant differences. Values are rneans * standard
error.
Acknowledggmenta
We are grateful to Dr. R.C. Scarpulla (Northwestem University) for the cytochrome
c promoter-CAT constructs, to Dr. J.C. McDermott (York University) for the PKC
expression vectors, to Dr. D. Nathans (Johns Hopkins University) for the Zif268 cDNA, to
Dr. V.P. Sukhatme (HarvardUniversity) for the EGR- 1 expression vectors, to Dr. G. Suske
(Philipps-Universitat Marburg) for the Spl expression vector. and to Dr. D.L. Barber
(University of Toronto) for the Raf expression vectors.
Results
PKC overexpression in L6E9 skeletal muscle cells.
L6E9 cells were CO-transfectedwith pRC4CATL326 along with various PKC
expression vectors or the control empty vector, pTB (Figure 1). Following differentiation,
the cells were treated with 0.75 p M A23 187 or DMSO.Overexpression of the Ca2'-sensitive
PKC isozyme, P,,, significantly increased the CAT activity over pTB. Overexpression of the
nPKC 6 isozyme did not significantly enhance the transactivation fiom pRC4CATi-326. An
unexpected fmding was obtained with the overexpression of the atypical PKC C. A23 i 87
treatment appeared to activate PKC 5,yielding CAT activities similar to those obsewed with
PKC a and Pl,. This was supnsing since the aPKCs do not require Ca2' for activation, as do
the cPKCs. However, there is some evidence that Ca2' can indirectly activate the PKCs via
the Ca2*-sensitiveprotease. calpain (42).
Co-rransfecrion of PKC and Raf expression vectors.
Transient transfections of wild type (Raf-1) and a dominant-negative Raf mutant
(Raf3Ol) were employed to detennine if Raf was acting downstream of PKC in the
transcriptional activation of cytochrome c observed in response to A23 187 treatment. The
dominant negative Raf3Ol reduced the transcriptional activation of cytochrome c mediated
by PKC a by 3 5 I 10% (Figure 2). However, RaBO l had no effect when transfected with
PKC 5 (Figure 3).
Protein Kinase C Isoforms
Figure 1 : Effect of owrexpressing mrious PKC isoforms on cytochrome c
transcriptional activation. PKC pIland 5 significantly increascd the CAT activity
compared to pTE3 ( ~ 3 - 6 )Bars
. represent the means f S.E.
"
PKCa
PKCa
PKCa
f
+
Raf- 1
Raf3 O I
Figure 2: Co-transfection o f PKCa with wild type or dominant negative Raf-I .
PKC a and R a f 3 O 1 CO-transfectionsresulted in a significantly l o w r transcriptional
actikation from pRCJCAT1-326 than with PKC a alone (n=4-6).
Figure 3: Co-transfection of PKCS with wild type or dominant negatiis
Rd-l (Ra130 1 ). No ciifferences wre obsened in transcriptional actim~ion
from pRC4CATI-326 witb PKC 5 donc or in combination with the Raf
consvucts ( I F 1-3).
MEK is invoIved in ,423 187-mediated increases in cytochrome c transcriptional activution.
The role of the dual-specificity kinase, MEK, was examined by the use of the MEK
inhibitor PD98059 (Figure 4).
L6E9 cells were pretreated with PD98059 at a final
concentration of 12.5 FM for 30 minutes pnor to the addition of A23 187. The pretreatment
with PD98059 resulted in a 37
15% reduction in transcription fiom pRC4CATb326 (n=5).
ERK I and 2 are acrivated in responre ru A23 187 treatment.
The ERK MAPKs are activated by MEK (12). Western blot analyses with a
phospho-specific anti-MAPK antibody were used to assess the activation of the ERKs in
response to A23 187 in L6E9 cells (Figure 5). A23 187 treatment resulted in a rapid and
transient activation of ERK 1 and 2, which peaked at 2 hours and diminished therafter. The
results of the experiments in which MEK was inhibited suggest that the rapid activation of
the ERK MAPKs plays some role in the transcriptional activation of cytochrorne c seen 48
hours following A23 187 treatment.
fierexpression of Sp l inhibits rranscriplional activationfiom pRC4CA TL326
The presence of an Sp 1 site in the cytochrome c promoter led us to examine the effect
of overexpressing Sp 1 with and without A23 187 treatrnent (Figure 6). Sp l overexpression
in non-treated L6E9 cells appeared to suppress the transcriptional activation of cytochrome
c. The CAT activity of cells transfected with the Sp l expression vector was 44
i 7 % lower
than the CAT activity of cells transfected with an empty vector ( ~ 7 ) This
.
suppression of
CAT activity observed in cells transfected with the Spl expression vector was not apparent
Figure 4: The role of MEK in A23 187-rnediated cytochrome c transactivation.
Pretreatment with the MEK inhibitor, PD98059,significantly reduced
the transcriptional activation from pRC4CATI-326 ( ~ 5 )Bars
. represent the
means c S.E. A representative autoradiograph of the CAT assays is inset. D=DMSO,
A=A23 187.
NT=Not treated
D =DMSO
A =A23187
PD=PD98059
Figure 5: Time course of MAPK activation in response to A23 187. A
representative western blot probing for phosphorylated ERK 1 and ERKZ.
ERK 1 @44), ERK2 @42).A23 1 87 transiently activates the ERK MAPKs.
Pretreatment with PD98059 completely abolished ERK activation .
EV=Empty Vector
A= A23187
Figure 6: Overexpression of Sp 1 inhibits the transcriptional activation
of cytochrome c. Spl overexpression resulted in a 44 k 7% reduction
in CAT activity compared to EV ( n=7). A23 187 treatment appears to
relieve the suppression caused by Spl (n=6). Bars represent the
rneans f S.E.
when the cells were treated with A23 187. There were no differences in CAT activity fiom
pRC4CATl-326 between cells transfected with the Sp 1 expression vector or the empty vector
(n=6).
Overexpression of Z$?68/Egr- 1 increases tramcripiional activation frorn pRClCA T/-326
Nt a culcium-dependent manner. independent of DNA binding.
Transient transfections of the wild type Egr- 1 (Egr- 1 930) or a mutant form incapable
of binding DNA @gr- 1 858) (39) were employed to assess the role of Zif268fEgr- 1 in the
transcriptional activation of cytochrome c (Figure 7). Neither the wild type, nor the mutant
form of Egr-1 altered the transcription fiom pRC4CATI-326 when treated with DMSO.
However, A23 187 treatment significantly increased transcripdonal activation in cells
ûansfected with Egr-1 858 and Egr-1 930, by 2.6-and 2.7-fold, respectively @<0.0 1, n=5).
The enhancement of transcriptional activation was approxirnately 32 5 9% greater than in
the presence of A23 187 alone.
Zif268 mRNA, but not protein, is induced by A23 187 treafmenf.
Steady-state levels of Zif268 mRNA increased 2.3-fold by 1 hour after the addition
of A23 187 (Figure 8). The mRNA levels remained elevated at 2 hours, and ihen declined
to non-detectable levels by 12 hours (data not show). The Uicreases in Zia68 mRNA were
reduced by 58% when pretreated with PD98059. Despite large increases in rnRNA, no
changes in Zif268 protein levels were detected by western blotting (Figure 9).
37
Figure 7: Overexpression of Egr-l 858 (mutant) and 930 (wild type)
significantly enhance the transcriptional activation of cytochrome c
in a calcium-dependent manner . -326/CAT+858+A and -326/CAT+930+A
are significantly greater than -326/CAT+858 and -326/CAT+930,
respectively @<0.05, n=3). Bars represent the means S.E. A=A23 187.
+
Time ( h )
1
-
2
4
Time (hours)
Figure 8: The effect of A23 187 on Zif268 mRNA. A23 187 treatment
induced a 2.3-fold increase in steady-state levels of zif268 mRNA by
1 hour. n i e increase seen at 2 hours was reduced by 58% by
pretreatment with PD98059 (n=2-3). Bars represent the means 2 S.E.
A representative Northem blot of Zif268 is shown above. Al1 blots
were striped and reprobed with 18s to correct for loading.
NT=Not treated
D =DMSO
A =A23187
62
--
N T-D A D A
Ihr
4hr
D A D A
24hr
48hr
Figure 9: The effect of A23 187 on Zif268 protein levels.
A23 187 did not affect protein levels of ZifL68, despite large
increases in steady-state mRNA levels. This is representative of
2 separate blots.
A23 187 treatment reduces the steady-stare levels of giutamafedehydogenme (GDM mRNA.
In order to evaluate potential downstrearn efTects of Zif26WEgr- 1 expression, we
measured GDH mRNA levels in cells treated with A23187, since the GDH promoter
contains Zif268Egr- 1 binding sites. Treatrnent of L6E9 cells with A23 187 at a final
concentration of 0.75 FMfor 12,24,36, and 48 hours resulted in a decline in the steady-state
levels of GDH mRNA (Figure 10). The mRNA levels began to decline at 36 hours of
treatment, such that by 48 hours, the GDH mRNA levels were 28 i 2% lower compared to
the levels observed at 24 hours (p<0.05, n=3-6). DMSO treatment had no effect on GDH
mRNA levels at any time point (Figure 11).
Time (hours)
Figure 10: DMSO treatment has no effect on
steady-state levels of GDH mRNA ( ~ 3 . )
ïime (hours)
Figure 1 1: A23 1 87-treatment reduces steady-state GDH
mRNA levels. GDH mRNA levels were 28 f 2% lower by
48 hours, compared to the levels observed at 24 hours
(~3-6).
Discussion
Contractile activity is known to stimulate mitochondrial biogenesis (49, however,
the specific signals involved are not lcnown. One possible second messenger is Ca"
.
Elevations in cytosolic Ca2' levels have k e n associated with increases in the activity of
mitochondrial enzymes (38,80),and alterations in skeletd muscle-specific gene expression
(1 6, 37, 56). We have previously s h o w that Ca" signals, mediated by A23 187, increased
steady-state mRNA levels of cytochrome c, malate dehydrogenase (MDH)and the P-subunit
of the F,-ATPase by 1.7-, 2.0-, and 1.9-fold, respectively. MDH enzyme activity was also
significantly increased by A23 187 treatrnent (38). In contrast, mRNA levels of cytochrome
oxidase IV and VIc did not change, while mRNA levels of the mitochondrially-encoded
cytochrome c oxi&se subunit Ili declined in response to A23 187 treatment (37). These data
suggest that Ca" signals differentially affect the expression of mitochondrial proteins denved
fiom the nuclear and mitochondrial genomes.
Using cytochrome c as a mode1 nuclear-encoded gene, we also showed that the
increase in cytochrome c mRNA could be attnbuted to transcnptional activation (38).
Further, we demonstrated that PKC was involved in the A23187-mediated increase in
transcriptional activation of cytochrome c, however, the specific isoform or isoforms
involved were not known. In the present work, we investigated this Ca" signahg pathway
M e r . To evaluate which of the different classes of PKC isoforms was involved in
cytochmme c transcriptional activation, we transfected L6E9 cells with expression vectors
43
encoding the a, P,,, 6, and C PKC isoforms. The a and P,, isofoms are conventional, Ca2+sensitive PKCs, while the 6 and C are novel and atypical PKCs, respectively, both of which
are Ca2+-insensitive.The results indicate that P,, and C isoforms were capable of enhancing
the transcriptional activation of cytochrome c in response to A23 187 treatment. This is an
expected resuit with respect to the P,, isoform, however we are uncertain about how Ca" is
activating PKC 5. One possibility is the involvernent of Ca" qactivated proteases, such as
calpain, since some PKC isoforms are substrates for calpain (53), however this is merely
speculative in the case of PKC
C. Nonetheless, Our data support the fmding that a PKC-
MAPK pathway is involved, since members of the conventional, novel, and atypical PKCs
cm activate the ERK MAPKs through MEK (79).
We continued to characterize the downstream components based on the mode1
proposed involving the following sequential activation steps: PKC a
- c-Raf - MEK -
MAPK (55). Through the use o f a dominant-negative Raf expression vector (RaBO 1). the
MEK inhibitor PD98059,and a phospho-specific anti-ERK antibody, we examined the
signaling pathway which led to the transcriptional activation of cytochrome c. The pathway
appears to be the same as that shown above for PKC a, since CO-transfectionof the dominant
negative mutant of Raf (RaBO1) partially blocked the A23 187 effect. Overexpression of
wild type Raf-l did not enhance the increase transcriptional activation of cytochrome c,
suggesting that M l is not a limiting factor in the signaling pathway. On the other hand, the
effect of PKC
C
on cytochrome c transcriptional activation was not affected by
CO-
îransfection of Rd30 1. This observation was also in agreement with the work of others, since
it has been demonstrated that PKC C actives MEK independently of Raf (79). However, our
efforts to block transmission of the calcium-activated signal with Raf3O 1 or PD98059 were
only partially effective. This strongly suggests that there are other, parallel, signaling
pathways also resulting in increased transcriptional activation of cytochrome c in response
to calcium signals.
We also sought to identify which transcription factors were activated to upregulate
cytochrome c transcription in response to elevated cellular Ca2+levels. This is a potentially
complex question, however, our work with cytochrome c promoter deletion constructs
indicated that the Ca2*-responsiveelement was mapped to a relatively short stretch of the
promoter, since the fold increases in CAT activity due to A23 187 treatment were similar
using the full length promoter (pRC4CATi-726), as well as the minimal promoter
@RC4CAT/-66). The minimal promoter contains consensus binding sites for both Sp 1 and
Zif268/Egr- 1 (see Figure 1 2). Therefore, we examined these, since Sp l and ZiR68iEgr- 1
can compete with each other for binding to GC-rich elements (46,52), or they can f o m a
complex and fiction cooperatively to upregulate transcription (60).
One way to examine the role of a transcription factor is to overexpress it and measure
its effects on transcription fiom the promoter fûsed to a reporter gene. The results of
overexpressing Sp 1 suggests that it may function to keep the transcription of cytochrome
c at a low level, since we observed a decrease in cytochrome c transcription. A33187
treatment appears to overcome this suppression. However, the effect of calcium on Spl
activity is not clear to us at the present tirne. Spl activity is regulated by post-translational
45
cgtagaam tcttttcaca atatggacag -P
atcta-gt
NF-AT
NF-AT
a t c c t c t a a t m t c c f gtttcttc cttaaagcn gcctgcacac cctccttgtt
NF-AT
ttttctacaa actaggggaa gaaaacaggg ataagatccc gtctgccaca tgtaccgcac
catccccagc tttgaggctt tcactgggag aacagaagat ggtttaacag agtgaaggtc
accagcatga gtccggtgca gttacgctag tcagggaatc tagaccaagc ctgggtaaag
gtcgccttca gactgggcgg cggrtcttag cactacc-aggc
agaaggagtc
Egr- 1
ctaagagccg gtgttacctg agcccagccg cacccaaatc ccpape~~aefccaccccg
Egr-l
ctactcgctc ctccccaaca cgcaggccgg a g g m t c c a c g t c cacgccttac
CRE
gtccaagggc ctptcgtaag tgtcgggcaa acgaggcctc tagaggaagg gcgccctctc
ggtacaacct accatgctag
- c
cacctt gctagccgc Ccaatcctgg
NRF- 1
CAAT
aeccaafaac atgcggctac gtcacggcgc agtgcccggc gctgccgcac gtccggccgç
CAAT
m
a g aacaagtgtg gttgcattga caccggtaca t a g p c n c p n e c g t g t c
Egr- 1
Egr- 1
cttgggctag agagcgggac gtctccc-tc
cmgtggtg t t g a c c ~
gcctgaccta caaagacatg cggaac
Figure 12: The full length promoter of the rat somatic cytochrome c promoter. Binding sites for a number of
transcription factors are indicated. C 1=CCAAT-like element (58).
modifications such as glycosylation and phosphoryiation (48), but we are not aware of any
data which suggests that calcium regulates Spl function. There is some evidence which
suggests a role for PKC in upregulating the expression of Spl (22), however this seems to
be cell-type specific (52).
Overexpression of Zif2681Egr-1 enhanced the transcription of cytochrome c in
response to elevations in Ca" levels, however. DNA binding did not appear to be required.
A similar fuiding was reported while studying Sp 1 (2 1). It was demonstrated that
CO-
ûansfection of a mutant fonn of Sp I incapable of binding DNA, along with wild type Sp I ,
upregulated transcriptional activation of a heterologous promoter in Drosophila SL2 cells
(2 1 ). This was attributed to protein-protein interactions between the mutant and wild type
Sp 1 proteins. We hypothesize that protein-protein interactions between Sp 1 and Zif268/Egr- 1
may account for the observed effects of Spl and Zif268i'Eg-r-1 on cytochrome c
transcriptional activation. in support of this, recent work has demonstrated that Spl and
Zia68 do interact with each other and alter gene transcription (4 1,60). Another possibility
is that Zif268lEgr-1 is sequestering another factor, such as Sp 1, thereby preventing it fiom
binding DNA. Previous work has demonstrated that Spl and Zif268tEgr- 1 can interact
regardless of whether or not one is bound to DNA (60),which suggests that sequestration
may be involved in the regulating cytochrome c transcription in response to overexpression
of these factors.
The absence of increases in the levels of Zif268Eg-1 protein in response to elevated
cytosolic Ca" levels is in agreement with other findings which indicate that Zif26WEgr-1
47
expression is regulated at the level of translation in cultured fibroblasts and muscle cells
(14,63). Although translational regulation is important, forced expression of Zi£268/Egr-1
does not appear sufficient to regulate cytochrorne c transcriptional activation. Posttranslational modifications, such as phosphorylation, have also been implicated in altering
the function of Zif268lEgr-1 (47). This seems consistent with OUT observations regarding the
effect of Zifï68Egr- 1 on the transcriptional activation of cytochrome c in cells treated with
A23 187, since there was no effect of Zif268Egr- 1 in vehicle-treated cells.
Although this work has focused on the transcriptional regulation of cytochrorne c in
response to elevated cytosolic calcium. the broader issue is the effect of calcium on the
mitochondrial phenotype. Our previous work, along with Our observations on GDH
expression (Figures 10, 1 1) contribute to the hypothesis that cytosolic calcium signals are
capable of altenng mitochondnal composition by changing the patterns of expression of
genes encoding mitochondnal proteins. In addition, since GDH is a potential target
of Zif268/Egr-1, our work with GDH suggests that Zif268Egr-1 is a potential factor
regulating mitochondrial phenotype. Elevated ~ a levels
"
increased the steady-state mRNA
levels of a number of genes encoding mitochondrial proteins, which suggests a switch to a
more oxidative muscle fibre (38). Future work examining the mRNA and protein levels of
a number of oxidative enzymes in response to elevations in ~ a levels
"
are necessary to
clarify the role of this cation in the 'metabolic' transformation of the skeletal muscle
phenotype. In view of the myriad of changes in gene expression in response to Ca" signals,
it is also important to detennine the physiological significance of these changes. Although
~ a alters
"
mitochondrial composition, the functional benefits remain to be resolved. This
could be addressed by rneasuring rates of respiration in mitochociria isolated from ionophoretreated cells.
Ln summary, Ca2' signaling has been the subject of extensive investigation. This
thesis considers the effect of calcium signais on the transcription of nuclear genes encoding
mitochondrial proteins. Our laboratory has employed a cell culture system in which
intracellular calcium concentration is increased by the use of the calcium ionophore, A23 187.
The results demonstrate that nudear genes encoding mitochondrial proteins are
differentially regulated by calcium. We attempted to dissect the signaling pathway leading
to enhanced transcriptional regulation of cytochrome c in response to elevated cytosolic ~ a " .
We have dernonstrated that a PKC-ER.MAPK pathway is involved, however, other
signaling pathways rnay play a role. since we were unable to completely block the
transcriptional activation of cytochrome c. Regulation of transcription of cytochrome c
seems to entai1 both Sp 1 and Zif268iEgr-1. DNA bindîng and immunoprecipitation assays
will be useful in understanding the interactions between these two factors, and how they are
altered by Ca" signais.
Future Work
1.
Electrophoretic mobility shift assays and antibody supershifis using segments of the
cytochrome c promoter should enable the identification of the factors which are
binding to the promoter, and how these factors are afTected by calcium signals.
2.
The use of site-directed mutagenesis of the cytochrome c promoter will allow us to
precisely identify the cis-elements that are Ca2+-responsive.
3.
Immunoprecipitation and immunoblotting will provide information on the
interaction between Sp 1 and Zif268/Egr- 1, and how such interactions are ahdty
calcium signals.
4.
The use of a protein kinase inhibitor, such as staurosporine, to determine whether
the the effect of A23 1 87 on Zif268fEgr- 1 (Figure 7) is the result of phosphorylation
of Zif268/Egr-1. Altematively, we could immunoprecipitate Zif268/Egr- 1 fiom cells
treated with A23187 and probe for phosphorylated forms with the use of a
phosphotyrosine or phosphoserine antibody.
5.
The use of phosphatase inhibitors would allow an evaluation of the role of
protein phosphatases, such as calcineurin, in the transcriptional activation of
cytochrome c.
-
Appendix A Corrected Data and Statistics
Table 1 : The CAT activity values obtained in CO-transfectionexperiments with the various
PKC expression vectors, expressed as the ratio of A23 187lDMSO (Figure 1).
r
i
PKC PH
PKC 6
PKC C
1.90
3.60
1.85
3.14
2.00
2.50
2.23
2.5 3
2.93
1.38
1.53
2.82
1.74
3.54
1.20
2.16
3.50
PTB
PKC a
1.13
2.00
2.40
L
Mean
1.42
2.10
3 .O3
2.04
2.90
S.E.
O. 19
0.17
0.32
0.24
0.32
Analysis of Variance: [F(4,15)=6.69,
p=0.003]
Table 2: Raw data, mean, and standard enor values for PKC and Raf CO-transfections
(Figures 2 and 3).
C
PKC a
a+Raf-1
a+Raf301 PKC C
c+Raf-1
C+
Raf30 1
-
2.70
1.30
1.90
3 .O0
4.20
2.60
1.50
2.10
2.80
2.50
1.50
2.40
2.00
1.50
0.70
1.20
1.60
1.90
7.-7
0.80
-
2.30
L.
r
Mean
2.65
2.2 1
1.72
1.43
2.1 3
S.E.
O. 14
0.4 1
0.28
0.40
0.48
Table 3: Student's t-test values.
t,
PKC a vs. a + Raf301
df
2.72, 7
p value
0.029
Table 4: Raw data, and statistical analysis of the role of MEK inhibitor on cytochrome c
transcriptional regdation (Figure 4.
CAT activity
CAT Activity +
PD98059
Standard error
2.37
1.94
3.75
1.25
1 0.31
1 0.24
I
t,
1 DMSO vs. PD98059
df
1 2.37
p value
1 0.04
Table 5: Raw data for the effect of overexpression of Spl on transcriptional activation of
cytochrome c (Figure 6).
Empty Vector
DMSO
1 S.E.
Analysis of Variance: [F(3,Z)=3-89, p=0.022]
Empty Vector
A23 187
1
Spl
A23187
Table 6: Raw data and for overexpression of Zif268/Egr-1 (Figure 7).
1"
-326 CAT
S.E.
-326CAT -326 CAT
Egr-1858 Egr-1930
DMSO
DMSO
1 0.45
Analysis of Variance: [F(1,76)=3 1.53, p=0.000007]
-326CAT
A23187
-326CAT
Egr-1 858
A23187
-326CGT
Egr-1 930
A23187
Table 7: Raw data and statistical analysis for zif268 mRNA measurements (Figure 8).
Time (h)
1
2
2 + PD98059
4
4 + PD98059
I
,
1.74
1.92
0.94
1.22
2.90
2.38
1.22
1.52
2.76
Mean
2.32
2.35
1.55
1 .O8
0.24
S.E.
0.67
1.43
0.10
Table 8: Raw data and stûtistical analysis for GDH mRNA measurements fiom DMSO
treated cells (Figure 10).
Time (h)
No
12
24
36
48
1 1.55
10.68
16.29
14.25
12.06
16.3 1
16.78
22.05
15.46
18.76
1 1.35
14.19
12.89
17.39
treatmen t
Mean
13.93
12.94
17.5 1
14.20
16.07
S.E.
2.38
1.93
2.35
0.74
2.04
Table 9: GDH mRNA expressed as a ratio of A23 187/DMSO (Figure 11).
Time (h)
-
12
24
36
48
1.10
1.26
0.85
0.92
0.92
0.90
1.12
0.88
- -.
-
-
Mean
1.O3
1.18
0.9 1
0.85
S.E.
0.05
0.14
0.1 1
0.02
Analysis of Variance: [F(3,11)=368,p=0.04]
Appendix B - Materials and Methods
RNA isolation from cetls
Protocol adapted fiom Anal. Biochem. 162: 156-159, 1987 and Am. J Physiol. 256: C 1092Cl 096, 1989.
Solution D (Denaturing solution)
4 M Guanidinium Thiocyanate
25 m M Nacitrate @H 7.0)
0.5% N-LauroyI Sarcosine
0.1 M 2-Mercaptoethanol added on day of experiment
Phenol (Nucleic acid grade) + 0.1% (wlv) 8-hydroxyquinoline, pH 7.6
Wash the cells twice using 5 ml cold PBS (no EDTA).
Add 3 ml of cold PBS and scrape the cells. Add to a 15 ml Falcon tube.
Spin in a clinical centrifuge for 2 min at 1000 rpm. Discard the supemate.
Add 1 ml of RNA extraction solution. Mix thoroughly.
Transfer to sterile eppendorf tubes containing 0.2 ml chlorofonn/isoamylalcohol
(24: 1) per 1 ml of RNA extraction solution used.
Shake vigorously for 15 sec and leave at room temperature 2-3 min.
Spin the samples in a rnicrofuge for 15 min at 4°C.
Transfer the upper phase carefblly to a new tube. Add 0.5 ml isopropanol per ml of
RNA extraction solution used to precipitate the RNA.
incubate at room temperature at least for 10 min.
Spin in a rnicrofuge for 10 min at 4°C. RNA forms a gel-like pellet on the bottom of
the tube.
Remove the supemate.
Add 1 mi of 75% ethanol per ml of RNA extraction solution used and wash the RNA
pellet by pipetting up and down a few times.
Spin in a microfuge for 1 min at 4°C.
Remove the supemate as best as possible and dry the pellet with a vacuum dessicator
for 5- 10 min. Do not let the pellet dry completely since the solubility will be low and
the A,JA,,
ratio will be lower than 1.6.
Whole ce11 extracts for western blot
1.
2.
3.
4.
5.
6.
Wash ce11 monolayer with phosphate-buffered saline (PBS)3 times. Aspirate last
wash.
Add 200 pl of 2x Laemmli buffer (62.5mMTris-HCl, pH 6.8, 20% glycerol, 2%
SDS,5% P-mercaptoethanol) with protease inhibitors.
S a p e surface of plate with mbber 'policeman' and place into 1.5 ml eppendodtube.
Sonicate sarnples on ice (3x1 0 second pulses).
Denature extract at 9S°C for 5 minutes. Spin (30 seconds) in microfuge to pellet
undissolved material.
Detemine protein concentration by Bradford Assay.
SDS Gel Electrophoresis
Adapted fkom Laemmli, U.K. Nature 127:680-5, 1970
Polyacrylamide Solution
30% (w/v) Acrylarnide
0.8% (wlv) Bisacrylamide
Filter through filter paper
Over Tris Buffer
1M Tris-HCI
add a sufficient arnount of bromphenol blue to get a light blue colour.
pH 6.8
Lysis Buffer
10Y0(vlv) glycerine
2.3% (wlv) SDS
62.5 rnM Tns-HCI (pH6.8)
Add 5% 2-Mercaptoethanol just pnor to use.
Electrophoresis Buffer
25.0 rnM Tris
192.0 m M Glycine
0.1%(wiv) SDS
pH to 8.3
Sample Dye
40% w/v sucrose in electrophoresis buffer
add a sufficient amount of bromphenol blue to get a dark blue colour.
store at -20°C
Separating gel (8% acrylarnide)
9.3 ml acrylamide solution ( I l . 7 ml for 10% gel)
14.45 ml &,O (1 2.05 ml for 10% gel)
1 1.25 ml 1 M Tris (pH 8.8)
0.3 ml 10% SDS
0.3 ml 10% APS
0.02 5 ml TEMED
Stacking gel (3% acrylamide)
1 ml acrylarnide solution
1-25 ml above Tris buffer
7.5 ml dHzO
0.10 ml 10% APS
0.10 ml 10% SDS
0.0 1 5 ml TEMED
Butanol overlay
12.5 ml 1.5 M Tris (pH 8.8)
0.5 ml 10% (w/v) SDS
volume up to 150 ml with dH,O
rnix the above solution 1:1 with Butanol
A two-phasic solution forms: the upper phase is butanol
1.
2.
3.
4.
Prepare separating gel. pour into plates with spacers, cover with butanol overlay,
allow to polymerize for 30 minutes.
Pour off butanol overlay, position comb, rnix and add stacking gel. Allow to
polymerize for 15 minutes.
Position gel in chamber with Electrophoresis buffer at the top and bottom. Remove
comb. Load samples.
Run gel at 100- 1 50 volts until dye fiont approaches the boaom of the gel.
Western BIot
Adapted from Maniatis, p.387-393.
Reaeents
Transfer Buffer
0.025 M Tris-HC1
0.15 M glycine
20 % (v/v) methanol
pH to 8.3
10X PBS
80 g NaCl
2 g KH,P04
35 g Na,HP04
2 gKC1
pH to 7.4
Volume up to 1 L.
5% Milk
5 g of Carnation skim milk powder
Volume up to 100 ml with 1X PBS
1% Milk
5% milk powder diluted with 1X PBS
Primary Antibody
1 :1000 dilutions in 1 % milk
Secondary Antibody
1: 1000-2000 dilutions in 1% milk
Detection
ECL Kit (Amersham)
ECL Hyperfilm (Amersharn)
Tramfer
1.
2.
3.
4.
5.
6.
Lay three sheets of the cut filter paper, soaked in tramfer bufTer, on top of the positive
electrode. Be sure that al1 edges match, and al1 corners are square.
Lay the nitrocellulose membrane, soaked in transfer buffer, on top of the stack.
Rime the gel with transfer bufTer to remove any particles, then position the gel on top
of the membrane.
Lay three sheets of filter paper, soaked in transfer buffer on top of the gel slab. Roll
glass rod over stack to remove any remaining bubbles.
Position the negative electrode onto the completed stack.
Transferat1.7mAlcm2for1hours
Rinse the membrane in 1X PBS.
Stain with Ponceau S for 1 minute to ensure even transfer.
Rinse for 10 minutes with 1X PBS to remove Ponceau S.
On shaker, block with 50 ml of 5% milk for 1 hou.
Wash with 1X PBS for 20 minutes.
Incubate with pnmary antibody at 4OC overnight.
Wash membrane twice for 5 min with 1X PBS/O.OS% Tween.
Wash for 20 min with 1X PBS.
incubate with secondary antibody for I hour at room temperature.
Wash 2 x 5 min with PBS/O.O5% Tween.
Wash for 20 min with 1X PBS.
Incubate membrane with ECL reagents for 1 minute and expose to film.
Quanti@ with laser densitometer.
DNA Transfection of L6 Cells
r
r
r
b
Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% FBS + 1%
pencillin/streptomycin
DNA (CAT constnict, P-gal constnict and carrier DNA)
1000x poly-L-ornithine (PLO)
dimethyl sulfoxide (DMSO)
For each plate prepare 3 ml of DNAlPLO solution as follows:
3ml DMEM (10% FBS, 1%PIS)
10-20pg of DNA of interest + 2pg of P-gal construct
3p1 of 1OOOx P L 0
DNA is added to the medium and mixed thoroughly before the addition of 1OOOx
PLO. Batch prepare DNAPLO solution to minimize differences between identical
plates.
Transfection is done when the cells are -60-70% confluent at the myoblast stage.
Remove the plates from the Uicubator and discard the medium fiom each plaie.
Add 3ml of DNAIPLO solution on the edge of each plate. Gently swirl the plate to
ensure uniforrn distribution of the DNAPLO solution during incubation. Retum
plates to the incubator.
Incubate the cells at 37'C for 6-8 hours. Ovemight incubation is also possible
(upto 12 hours).
DMSO shock is accomplished in the following marner. The medium is discarded
and replaced with 5ml of DMEM + 10% FBS + 1% P/S supplemented with 25%
DMSO.
Afier 2.5 minutes incubation at room temperature, cells are washed 3 times with
DPBS (pre-warmed to 37°C)
Add 8ml of DMEM supplemented with 5% HS + 1% PIS. Fresh media is added
every 48-36 hours.
Preparation of Chlorarnphenicol Acetyltransferase (CAT) extracts from cells
r
r
1.
2.
3.
4.
5.
6.
7.
Phosphate buffered saline (PBS)
0.25 M Tris-HCI, pH 7.9
Liquid nitrogen
Rinse celIs twice with cold PBS.
Add 1.2 ml of cold PBS to plate and scrape. Transfer to 1.5 ml eppendorf.
Spin at 4OC for 5 minutes.
Discard supernate, add 1 0 0 ~of
1 0.25 Tris-HC1 and vortex to disperse the pellet.
Imrnerse in liquid nitrogen for 1 minute.
Thaw at 37OC for 5 minutes. Vortex.
Repeat fieeze-thaw cycle 2 more times.
8.
9.
Spin for 5 minutes in microfuge at 4OC.
Transfer supernate to new tube and store at -20°C.
Chloramphenicol acetyltransferase (CAT) Assay
r
b
b
0.25 M Tns-HCI, pH 7.9
L6 extract
''C-chloramphenicol
Acetyl CoA, lithium salt
Ethyl acetate
Chloroform:Methanol(95 5 )
Thin Layer Chromatography plates
incubate extracts with "C-chloramphenicol and acetyl CoA for 2 hours at 37OC.
Extract chloramphenicol by adding 0.5 ml of ethyl acetate, vortex and spin for 30
seconds in a microfuge.
Transfer organic (upper) phase to 1.5 ml eppendod tube.
Evaporate the ethyl acetate in a speed-vac microfuge for 30 minutes.
Resuspend the pellet in 15-2011 of ethyl acetate.
Prepare the TLC chamber by adding 150 ml of chloroform:methanol(95:5) 1 hour
prior to the inserting the TLC plate into the chamber.
Apply the samples to the TLC plate with a pipette.
insert the TLC plate into the chamber and remove it once the solvent migrates to
the top of the plate (-20 minutes).
Quanti@ the acetylated and unacetylated derivatives by electronic autoradiography.
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