Handout in pdf

Sphingolipid metabolism and
function: implications to
cancer and aging
Lina M Obeid
Medical University of South Carolina
Charleston, S.C.

Overview
Sphingolipid metabolism
Sphingolipid function
Sphingolipid mediated biology
Cellular
senescence
Aging
Cancer
Telomerase Apoptosis
Angiogenesis

Sphingolipid Metabolism

Sphingolipid function
evidence for roles for sphingolipids in
signaling and cell regulation
A: Modular Action
– Regulated metabolism of sphingolipids
– Multiple bioactive sphingolipids; sphingosine, ceramide,
sphingosine-1 phosphate, and others.
– Identification of targets for sphingolipid action: mechanisms of
action e.g. kinases, phosphatases and receptors.
– Deciphering mechanisms of regulation of sphingolipid metabolism.
– Molecular genetics of sphingolipid function in yeast.
B: Other Functions
– Critical membrane components: required for viability in yeast and
mammalian cells.
– Enzymes of sphingolipid metabolism as targets for anti fungals and
antibiotics
– Cell cell interactions
– Receptors for various ligands and toxins

Cellular Senescence:
a model to study aging
Inverse correlation between age and life
span of cells in culture
Correlation between life span of species
and its cells in culture
Mortal cells have a finite life span in
culture (limited population doublings)

Biology of Cellular Senescence
Inability to proliferate
Inability to undergo DNA synthesis
Telomere shortening
Senescence is dominant
Resembles a state of terminal
differentiation

Ceramide Levels and Sphingomyelinase
Activity Increase in Senescent Cells

Ceramide Induces a Senescent Phenotype
in Human Diploid fibroblasts

Ceramide and cellular
senescence
Ceramide levels and SMase activity increase
when cells become senescent
Ceramide induces a senescent phenotype
morphologically
biochemically (inhibits cell cycle progression, inhibits
the PKC/PLD pathway, induces Rb dephosphorylation,
inhibits AP1 activity…)

Telomeres
The ends of eukaryotic chromo-somes
are capped by short G-rich sequences
(telomeres) that are tandemly
repeated for several hundred to
thousand base pairs.
In humans the telomeric repeat is
TTAGGG.
Telomeres prevent chromosomal
degradation, end-to-end fusion, and
chromosomal rearrangements.
Human telomeres shorten with each
cell division.
Telomeric shortening is thought to
function as a molecular clock that
triggers senescence and crisis.
Haber, D.A. NEJM 332 (14):955 , 1995

Background on Telomerase
Telomerase is a ribonucleoprotein enzyme
complex that adds telomeric repeats
(TTAGGG) n to the ends of chromosomes.
Telomerase activity is detected in malignant
cells, immortalized cells, and germ cells, and is
not detected in most somatic cells.
It has been shown to be over expressed in 85-
90% of human cancers.
It is not sufficient to cause neoplasia on its
own. Its presence enables malignant cells to
maintain telomere length, allowing infinite
replicative capacity.
Buys, CHCM. NEJM 342 (17):1282 (2000)
Increased telomerase activity has been
suggested to offer protection against apoptosis

The Human Telomerase Ribonucleoprotein Complex
The human telomerase complex
consists of:
• hTERT (the catalytic reverse
transcriptase subunit)
• hTR (the RNA template)
• Telomerase associated proteins:
TEP1 & p23/hsp90
• protein components of snoRNPs
including dyskerin
• DNA-binding proteins TRF-1 & TRF-2
• TRF-1 associated proteins TIN2 &
tankyrase
• other associated proteins
Estimated molecular mass > 1000 kDA
Shay, J.W. and Wright, W.E. Science 286;2284-2285 (1999).

Ceramide Inhibits Telomerase
Activity
OH
1 5
2
HO 3
4
N
H
(2S,3R)
18
16 '
O
D- erythro-C 16 -Ceramide
OH
1 5
2
HO 3
4
N
H
(2S,3R)
18
16 '
O
D- erythro-4,5-dihydro-C 16 -Ceramide

Ceramide causes a decrease in
telomerase mRNA

Ceramide inhibits telomerase
transcription

Ceramide mediates ubiquitination
and degradation of c-Myc

CONCLUSIONS
Ceramide is a possible upstream regulator of telomerase
Inhibition of telomerase by ceramide involves rapid proteolysis
of c-Myc transcription factor (by increased ubiquitination),
which decreases the hTERT promoter activity, leading to
decreased transcription and telomerase activity
Increased endogenous ceramide levels in response to various
stimuli such as chemotherapy might be a potential therapeutic
modality
Ceramide-induced telomerase inhibition and telomere length
reduction is important for growth inhibition and senescence
Ogretmen et al., J. Biol. Chem. 276:32506-32514, 2001

Role of Ceramide in Apoptosis
• A growing body of evidence is pointing to important roles of
ceramide in apoptosis.
• This evidence comes from studies showing:
1. Most apoptotic stimuli induce ceramide formation with
appropriate kinetics; multiple mechanisms.
2. Exogenous and endogenous ceramides are sufficient to
induce apoptosis
4. Endogenous ceramides are necessary
5. Cellular mechanisms are increasingly providing links
between ceramide metabolism, its action, and other key
components of apoptosis (bcl-x, bcl-2, upstream caspases,
downstream caspases, p53, ROI’s, mitochondria)
6. Yeast models of involvement of sphingolipids in stress
responses

Bax
Singomyelinase
Ceramide

Bax
Sphingomyelinase
Ceramide

Ceramide and Mitochondria
1. Activation of sphingomyelinase (SMase) is
sandwiched between the action of upstream
caspases and downstream caspases.
2. Exogenous ceramides specifically activate
downstream caspases and not upstream
caspases; also, ceramides induce cytochrome
c release.
3. Activation of SMase is dependent on
ROIs/drop in GSH.
4. New data on a more intimate connection.

Neutral sphingomyelinase activity in
MAMs from liver
Acid P-ase
umole/mg/hr
F 0
F 1
ATPase
umole/mg/hr
NSMase
nmol/mg/hr
Acid Smase
nmol/mg/hr
H
3.77
13.2
8.9
133.9
S
1.67
-
4.7
14.35
M
12.9
28.5
20.6
489
PM
5.2
35
8.24
295
MAM 2.6
18
23.5
111.7
Table 2: Enzyme assays in the different cellular fractions.
H = Homogenate fraction following low spin centrifugation (1000 g x 5’).
S = Post mitochondrial supernatant. M = crude mitochondria.
PM = pure mitochondria isolated following percoll gradient.
MAM = mitochondrial associated membranes.

Ceramide Levels in Liver
Mitochondria
200
B Ceramide
control
Ceramide pmoles/pi
150
100
50
37Þ C
bSMase
0
H S M PM MAM

Construction of bSMase-GFP vectors with targeting signals
to different intracellular compartments
pCMV/myc

Cellular localization of the bSMase-GFP fusion protein
in MCF7 cells
plasma membrane cytoplasm Mitochondria
Golgi ER Nuclei
GFP Mitotracker Overlay

Increase of SMase activity in MCF7 cells
transfected with the six bSMase-GFP vectors
Specific activity (nmol/h/mg)
6
4
2
0
PM-control
PM-bSMase
* * *
12 24 48 72 96
Time (h)
50
40
30
20
10
0
Cyto-control
Cyto-bSMase
*
* *
12 24 48 72 96
Time (h)
30
20
10
0
Mito-control
Mito-bSMase
*
*
12 24 48 72 96
Time (h)
Specific activity (nmol/h/mg)
40
30
20
10
0
Golgi-control
Golgi-bSMase
*
*
* *
12 24 48 72 96
Time (h)
600
400
200
0
ER-control
ER-bSMase
* *
* * *
12 24 48 72 96
Time (h)
40
30
20
10
0
Nucleus-control
Nucleus-bSMase
*
* *
12 24 48 72 96
Time (h)

Increase of ceramide levels in MCF7 cells transfected
with the bSMase-GFP constructs
PM-control
Cyto-control
Mito-control
Ceramide (% of control)
130
110
90
70
50
PM-bSMase
12 24 48 72
Time (h)
130
110
90
70
50
Cyto-bSMase
12 24 48 72
Time (h)
150
130
110
90
70
50
Mito-bSMase
* *
12 24 48 72
Time (h)
ER-control
Golgi-control
Nucleus-control
Ceramide (% of control)
300
250
200
150
100
50
ER-bSMase
*
*
*
12 24 48 72
Time (h)
200
150
100
50
Golgi-bSMase
*
*
12 24 48 72
Time (h)
150
125
100
75
50
Nucleus-bSMase
12 24 48 72
Time (h)

30
20
10
0
Mitochondrial targeting of the
bSMase induces cell death
A
30
Mito-control
Mito-bSMase
25
20
15
10
5
0
24 48 72
Time (h)
B
Dead cells
(% of total transfected cells)
PM-control
PM-bSMase
Cyto-control
Cyto-bSMase
Mito-control
Mito-bSMase
ER-control
ER-bSMase
Golgi-control
Golgi-bSMase
Nuc-control
Nuc-bSMase

verexpression of the Mito-bSMase-D295G mutant does
not cause elevation of SMase activity , ceramide levels
or cell death.
20
15
10
5
A B C
150
25
125
100
75
* *
20
15
10
Ceramide (% of control)
5
Dead cells
(% of total transfected cells)
*
Specific activity (nmol/h/mg)
control
Mito-bSMase
Mito-bSMase-D295G
Mito-control
Mito-bSMase
Mito-bSMase-D295G
Mito-control
Mito-bSMase
Mito-bSMase-D295G
0
50
0

Bax
Sphingomyelinase
Ceramide

Summery
Evidence for a mitochondrial pool of
ceramide
Evidence for a MAM pool of SMase
Only increases in mitochondrial
ceramide induce the apoptotic program

Sphingomyelin
Glycolipids
GCS
Senescence
SMase
CERAMIDE
+ +
CDase
Inhibition of Telomerase
SPH
Apoptosis
-
De novo synthesis
SPH-1-P
+
Proliferation
Migration
Angiogenesis

Acknowledgements
Coworkers
Cungui Mao
Ruijuan Xu
Helene Birbes
Jackie Kraveka
Korey Johnson
Heather Greer
Kashelle Othersen
Tarek Taha
Mark Venable
Collaborators
Yusuf Hannun
Besim Ogretmen
Alicia Bielawska
Zdzislaw Szulc
Julnar Usta
Administrative Support
Kathy Wiita
National Institutes of Aging
Veteran’s Administration Merit Award
Paul Beeson Physician Faculty in Aging Award

Web site
www.musc.edu/bcmb/ceramide