Transcript munoz - California State University, Los Angeles

Analysis of Id-1 and Twist-1 Regulation in
Bone Development
Anna E. Muñoz
Cal State University, Los Angeles-City of
Hope Cancer Collaborative
April 7, 2008
Outline
o Introduction
o Helix-Loop-Helix proteins
o Id-1 and Twist-1
o Human stem cells
o Study model system
o Significance of Study
o Cell line preliminary results
o Collaborative research project
o Project overview
o Acknowledgements
Helix Loop Helix Proteins
o Various helix-loop-helix (HLH) proteins play a key
role in the regulation of cellular growth and
differentiation
o Basic HLH, bHLH, proteins include a basic DNA
binding domain
o MyoD (directs muscle development) and
TWIST-1
o HLH proteins lack the basic domain
o Id proteins do not bind DNA
Id-1
o
o
o
o
o
Belongs to the Id protein family (Id-1, 2, 3, 4)
HLH protein
Inhibitor of differentiation
Preferentially dimerizes with bHLH proteins
Acts in a dominant negative fashion
o Prevents bHLH proteins from forming dimers with other bHLH
proteins
o Prevents bHLH proteins from binding DNA
o Is differentially regulated during differentiation of
mesenchymal stem cells to different cell types
Twist-1
o bHLH transcription factor
o Homodimer or heterodimer with other bHLH proteins
(i.e. E proteins)
o Regulates cell movement and mesoderm
development during early embryogenesis (i.e. bone
and muscle)
o Twist-1 has both positive and negative functions
regulating mesenchymal cell differentiation
o Binds to a conserved E-box sequence (CANNTG) on
the promoter region that activates or inhibits
transcription of a target gene
HLH Proteins
Id
bHLH
bHLH
+1
E-Box
Stem Cells
o Unspecialized
cells
o ability to self
regenerate
o ability to
differentiate into
other cells
http://stemcells.nih.gov/info/scireport/chapter5.asp
Mesenchymal Stem Cells
o Also known as “bone marrow stromal cells”
o Capacity to differentiate along myogenic, chondrogenic,
osteogenic, and adipogenic lineages
www.worldhealthspecialists.org/stemCellBasics.asp
Study’s Model System
o Normal human cells undergo a limited number of cell
divisions in culture
o Enter senescence, a non-dividing state
o Telomere shortening has been linked to cellular
senescence
o Retroviral transduction of human telomerase reverse
transcriptase (hTERT)
o Maintains telomere length
o Extends life span
o Dr. Glackin’s lab at COH created a human fetal
mesenchymal stem cell line that has been
immortalized by the hTERT gene, hfMSC-SK-hTERT cell
line
Mol Biol Cell, 2005, 16:1491-1499
Significance of Study
o Different members of the Id family are
overexpressed in different tumor types
o Abnormally high expression of Twist-1 in
cancer cells has been associated with
metastasis
o Invasive breast cancer
o Twist-1 overexpression prevents normal bone
and muscle development
o The molecular basis of mechanisms that
induce the differentiated osteoblastic
phenotype is poorly understood
CELL LINE PRELIMINARY RESULTS
Experimental Methods
o Cell culture experiment performed by Dr. Glackin in
2007
o To determine the expression of Id-1, Id-2, Twist-1,
Dermo-1 and bone markers during the differentiation
of hfMSC-SK-hTERT cell line to bone.
Experimental Methods
o Cells were grown in expansion medium
o Alpha-Minimal Essential Medium supplemented with Fetal
bovine serum, penicillin, streptomycin, L-glutamine, and
ascorbic acid 2- phosphate.
o Differentiation was induced by changing medium
conditions.
o Expansion medium was supplemented with
dexamethasone, sodium pyruvate, hepes, and inorganic
phosphate to induce differentiation to bone.
o Differentiation was carried out for 28 days
o RNA was collected at days 2, 4, 7, 14, 21, and 28
o Expression of the genes listed above along with
bone marker genes was measured by real time RT
PCR
Experimental Methods
DAY 4
DAY 2
OSTEOGENIC
ADIPOGENIC
MYOGENIC
OSTEOGENIC
ADIPOGENIC
DAY 7
MYOGENIC
OSTEOGENIC
ADIPOGENIC
MYOGENIC
Diff
Media
Control
Media
DAY 14
OSTEOGENIC
Diff
Media
Control
Media
ADIPOGENIC
DAY 21
MYOGENIC
OSTEOGENIC
ADIPOGENIC
DAY 28
MYOGENIC
OSTEOGENIC
ADIPOGENIC
MYOGENIC
MSC differentiation to bone
hfMSC Osteogenic Timecourse
35
30
25
Fold Change
20
15
10
5
0
-5
-10
Day2
Day 4
Week
1
Week
2
Week
3
Week
4
Unpublished preliminary data collected by Dr. Glackin, 2007
Twist-1 and Id-1 Expression in Osteogenic
Differentiation of MSCs
Id-1
TwitstMSC
Osteoprogenitor
1
Preosteoblast
Osteocyte
Osteoblast
Bone Cell Lining
CANCER COLLABORATIVE
RESEARCH PROJECT
Research Goals
o To compare the regulation of Id-1 and Twist-1 in the
hfMSC-hTERT cell line throughout its osteogenic
differentiation.
o To identify and analyze the regulatory features of
the Id-1 and Twist-1 promoters that contribute to the
development of MSCs to osteoblasts.
Id-1 and Twist-1 Regulation in MSC-hTERT line
o To compare Id-1 and Twist-1 regulation
Grow cells in
maintenance
medium
Maintain cells at 70%80% confluency
Change medium every
3 days
Obtain mRNA from cells
at various time points
Introduce osteogenic
medium to promote
differentiation
Perform Quantitative
PCR
Analyze Id-1 and
Twist-1 expression
Human Id-1 and Twist-1 promoter constructs
o Make Id-1 and Twist-1 promoter/reporter constructs
o To study transcriptional regulation of Twist-1 and Id-1
in differentiating MSCs
Bioinformatic analysis
of human Id-1 and
Twist-1
Isolate genomic DNA
from fhMSC-SK-hTERT
cells
Clone Id-1 and Twist-1
upstream regions via PCR
Design primers for
human Id-1 and Twist-1
upstream regions
Perform luciferase
assays
Grow cells and
differentiate
Grow and isolate
sufficient quantity
luciferase reporter vector
Ligate promoters
into vectors
Transform fhMSC-SKhTERT cells
Acknowledgements
o CSULA-COH Cancer Collaborative Program
o NIH grant
o Dr. Sharp, Cal State LA
o Laura Martinez, Sharp Lab
o Dr. Glackin, City of Hope
o Shan Li, Glackin Lab
o Joyce Ho, Cal State LA Collaborating student
Thank You