Presentation On Wnt 4 and rhe role of HDAC4 & SIRT1 in bone biology
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Transcript of Presentation On Wnt 4 and rhe role of HDAC4 & SIRT1 in bone biology
GAYATHRI VIJAYAKUMAR
Dr. Nicola C. PartridgeDept of Basic Science & Craniofacial BiologyNYU-CD
Wnt proteins are a family of 19 highly conserved cysteine-rich secreted glyco-lipoproteins (39-46 Kda) that regulate development, cell proliferation and motility, cell fate determination, generation of cell polarity and embryonic inductive interactions. They do this by regulating and stimulating proliferation along the Notch-Wnt signaling pathway.Wnt protiens grouped into canonical and non-canonical.Wnt molecules can bind to the receptors LRP5, Frizzleds, Ror2 and RYK at cell surfaces to activate intracellular signaling pathways.
WNT PROTEIN FAMILY:
TS19 mouse embryo with areas of Wnt signaling visible in red
Targeted knockout of the Wnt4 genes in a female mouse shows that the kidney fails to develop.
Wnt signals induce cancerous changes.
Wnt Signaling Pathway
Canonical
Non-canonical
B-catenin
Ca 2+ PCP
• Osteoblast cell proliferation•Cell fate determination•General bone maintenance•Body axis specification•Cancer
• intracellular Ca2+ conc. Activation of
JNK
Affect cytoskeletal organisation
B-catenin
Ca 2+PCP
WNT 4 (Wingless-type MMTV integration site family, member 4):
•Wnt-4 has the distinction of being the first signaling molecule known to influence sex-determination by participating in gonadal formation . This gene is essential for female development and for suppressing the male reproductive system. • As a mature molecule, human Wnt-4 is a 46 kDa, 329 aa glycoprotein that contains 24 cysteine residues and two possible N-linked glycosylation sites.• Wnt-4 expression in mesonephric and gonadal mesenchyme promotes the development of the Müllerian duct (a structure present in the embryo that develops into the uterus, fallopian tubes, cervix, and the upper part of the vagina) and blocks the development of Leydig cells.
• In the same region, Wnt-4 expressing mesenchyme induces a mesenchyme-to-epithelium transition that promotes nephron development.•In the embryonic adrenal cortex mesoderm, Wnt-4 expression contributes to the proper formation of the zona glomerulosa, a region that secretes aldosterone.• Wnt-4 is also found in virgin mammary epithelium. Progesterone increases Wnt-4 expression, leading to an enhanced side-branching (but not elongation) of mammary ducts. •Wnt-4 is detected in fibroblasts in areas of wound healing where fibrin degradation products are abundant.
The WNT4 gene is located on the short (p) arm of chromosome 1 between positions 36.23 and 35.1.
WNT4 – A PTH ANABOLIC MEDIATOR
Serum Ionized Ca2+ level decreases
Parathyroid Gland
PTH Release
PTH binds to PTHR-1 in Bone, Kidney and other target tissues
PKC
PKARegulates WNT4
Enhance Osteoblast
Differentiation
Through Non-canonical Wnt/Ca2+ and Wnt/PCP pathway
Cell Proliferation & CFU-
F numbers
By inhibiting BMSSCs apoptosis & stimulating their rate of growth
PTH induces WNT4 mRNA expression in osteoblastic cells 8 hours after PTH injection and maximum expression is observed in the mineralization phase.
Expressed in chondrocytes & mature osteobalsts
WNT4-AN ENHANCER OF STEM CELL OSTEOGENIC DIFFERENTIATION
Primary osteoblasts
Wnt 4
PCP & Cgmp/Ca2
+ Pathway
MouseBMSSCs
Wnt 4
B-catenin Pathway
Stimulates
osteocalcin mRNASome
control in osteoblast differentiati
on
Affects proliferation
& differentiati
on
WNT4 , a noncanonical member, was found to potently enhance osteogenic differentiation of MSCs isolated from craniofacial tissues in vitro and bone formation in vivo.
Interestingly, Wnt4 did not increase the cytosolic level of B-catenin. But activated a novel noncanonical signaling pathway, P38MAPK, which is known to positively regulate osteogenic differentiation induced by BMPs and other growth factors
Ca2+ Pathway
Proliferating mBMSSCs
Differentiating mBMSSCs
mBMSSCs
+ WNT4
Osteocalcin Bone Marker
gene expression
Adipocyte Marker Genes
Both Osteogenic &
non-osteogenic conditions
•In uncommitted MSCs, Wnt4 stimulates B-catenin pathway inducing proliferation.
•In committed osteoblasts, it stimulates non-canonical pathways responsible for its effect on differentiation.
•Duality of Wnt4’s action depends on 1. Development stage of the
cell2. Active proliferatio of the
cell
Specific AimsAim 1 : Assess the Wnt-4 effect on proliferation of human bone marrow stromal stem cells.Aim 2 : Assess the Wnt-4 effect on differentiation of human bone marrow stromal stem cells.
Experimental DesignControl and WNT-4 conditioned media collection:Nih3t3
Control Cells grown in DMEM supplemented with 10%FBS & 1%p/s
Medium collected @ 80% confluency
After syringe filtration
Nih3t3 Wnt4 Producing Cells grown in DMEM supplemented with 10%FBS & 1%p/s
Medium collected @ 80% confluency
After syringe filtration
Stored @-80C
Culturing Human BMSSCs with Recombinant WNT4
Human Cancellous Bone Specimen
Isolate hBMSSC
s
Plate cells at a conc of 1 million to 3 million cells/well in a 12 well plate
Medium: 10%FBS Alpha-MEM+ 1% p/s+1%Glu+Dex24ng/ml Wnt4 added to the wnt4 wells
Left untouched for 5 days for the cells to
adhere
Day 5: Medium changed to
contain 1 μg/ml Ascorbic Acid
Medium Changed every 2-3
days
Day 12: Viable Cell
Count & Picture Taken
Control hBMSSCS
hBMSSCs treated with Wnt4
SIRT1 (Class III HDACs) ProjectSirtuin 1, also known as NAD-dependent deacetylase sirtuin-1 stands for sirtuin (silent mating type information regulation 2 homolog) 1.Sirtuin 1 is a member of the sirtuin family of proteins, homologs of the Sir2 gene in S. cerevisiae.Sirt 1 is highly expressed in the foetal and adult brain .Sirt1 not only deacetylates histones H1, H3 and H4, but also deacetylates many non-histone proteins including p53, FOXO, Ku70, p300, Rb, E2F1, NF-kB, p73 and PGC-1α .In virtue of these important targets, SIRT1 is linked to regulatory control of diverse normal and abnormal cellular processes ranging from stress responses, aging, and metabolism to cancer.It is down-regulated in human senescent cells, suggesting that SIRT1 may be required to extend replicative life span It deacetylated p53,thus antagonizing p53 mediated apoptosis and increasing cell survival. Cancer cell lines revealed higher endogenous level of SIRT1 expression compared to normal cells .
HDAC4:Histone deacetylases (HDACs) are transcriptional coregulators with the capability of modifying chromatin structure and other transcription factors.HDACs play a role in deacetylation of lysine residues in the tails of core histones.They are classified into 4 groups. HDAC4 binding sites are three serine residues, which are calcium-calmodulin –dependent kinase (CaMK) phosphorylation sites.
C-Fos:c-Fos is a cellular proto-oncogene belonging to the immediate early gene family of transcription factors. c-Fos has a leucine-zipperDNA binding domain, and a transactivation domain at the C-terminus.
C-Jun:c-Jun is the name of a gene and protein that, in combination with c-Fos, forms the AP-1 early response transcription factor. It is activated through double phosphorylation by the JNK pathway but has also a phosphorylation-independent function.It is a proto-oncogene as well and belongs to the IEG family.
PTH Induced Bone Resorption & Bone Remodeling
PTH regulates calcium metabolism by acting on osteoblasts to produce Osteoclast Activating Factors such as RANKL, MMP-13, or Collagenase-3.PTH induces MMP-13 gene transcription through a PKA dependent pathway.
MMP-13 Promoter
RD AP-1
Runx2
HDAC-4
MMP-13 Promoter
RD AP-1
Runx2
HDAC-4
PKA dependent phosphorylation
PTH
Nucleus
p300
pcafHAT RD AP-
1
p300
pcafHAT
Runx2
JUN & FOS
Under Basal Condition
PTH Effect
Cytoplasm
MMP-13 Promoter
RD AP-1
Runx2
HDAC-4
JUN & FOS
p300
pcafHAT
Sirt-1
AIM:Investigating the role of Sirt-1 in regulating the expression of MMP-13.Establishing the characteristics of SIRT1 association with AP-1 proteins in PTH-treated Osteoblasts.Whether there is association of Sirt-1 with Fos or Jun in Osteoblastic cells.Whether there is an interaction between HDAC4 and Sirt1 as well as between HDAC4 , Fos and Jun.
EXPERIMENT:
UMR cells cultured till 80-90% confluent
PTH treatment for 4 and 8 hours
Preparation of cell lysate
Immunopercipitated for Sirt1, Fos, Jun or
HDAC4
Immunoblot for Sirt-1, HDAC4, Fos
or Jun
IP IgG
jun
Fos
Immunoblot Sirt-1
IP IgG
Sirt-1HDAC4
Immunoblot Sirt-1
C4 P4 C8 P8 C4 P4 C8 P8 C4 P4 C8 P8
C4 P4 C8 P8 C4 P4C8 P8 C4 P4 C8 P8
IP IgG
jun
Fos
Immunoblot HDAC4
IP IgG
Sirt-1HDAC4
Immunoblot HDAC4
C4 P4 C8 P8 C4 P4 C8 P8 C4 P4 C8 P8
C4 P4 C8 P8 C4 P4 C8 P8 C4 P4 C8 P8
THE ORTHOFIX PROJECT
PULSED ELECTROMAGNETIC FIELD THERAPY:
Pulsed Electromagnetic Field Therapy (PEMF), also called Pulsed Magnetic Therapy, is a reparative technique most commonly used in the field of orthopedics for the treatment of non-union fractures, failed fusions, and congenital pseudarthrosis.
HISTORY:In1970s,the FDA approved the use of a specific biphasic low frequency signal for the treatment of non-union/delayed fractures –introduced by the Andrew Basset team.A decade later, FDA allowed the use of pulsed radiofrequency electromagnetic field (PRF) for treatment of pain and edema in superficial soft tissues.
Three biological windows have been identified by analyzing the response of cells to a range of amplitudes and frequencies. These are:
bone unification
reduce pain
edema & inflammation
increase blood circulation
stimulate immunity
endocrine systems.
1. 50–100 lT (5–10 Gauss)2. 15–20 mT (150–200 Gauss) and3. 45–50 mT (450–500 Gauss) (Markov 2005).
PEMF
NASA Signal Driver (Hydra) Project Protocol
Rat Primary osteoblasts isolated from postnatal
day 1 rat calvariae
Plated with MEM+10% FBS. Treated with the PEMF signal 4h a day. Control not treated.
Day 6 (proliferation stage) : Cell number counted
Medium switch with BGJ + 10%FBS + Ascorbic Acid + ß-glycerophosphate
Day 8: Induction of differentiation
Day 12 & Day 18: Total RNA isolated from cells & von
Kossa Staining
RT-PCR
Genes Analyzed in the NASA-ORTHOFIX Project
1.Osteocalcinalso known as bone gamma-carboxyglutamic acid-containing protein (BGLAP), is a noncollagenous protein secreted solely by osteoblasts.It is used as a biomarker for bone formation
4.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH ) is an enzyme that catalyzes the sixth step of glycolysis besides transcription activation, initiation of apoptosis, and ER to Golgi vesicle shuttling. Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a housekeeping gene.
2.Bone Alkaline Phosphatase (BAP)It is secreted by osteoblasts. Alkaline Phosphatase (ALP) is a ubiquitous enzyme associated with cell membranes.. It is produced by osteoblasts to provide a high PO4 concentration at the osteoblast cell surface during bone mineralization and is a marker of bone formation.
3.Type I Collagenformed by osteoblasts; reflects rate of collagen and bone formation; most sensitive marker of bone formation and particularly useful for monitoring bone formation therapies and antiresorptive therapies.
Osteoblast Mineralization – von Kossa Staining
Day 12 &18
Cells fixed with 95%
Ethanol (15 min at 37°C)
Rinsed with 80%
Ethanol
Rinsed with 50% Ethanol
Rinsed with 20%
Ethanol
Washed with water
Incubated with 5% silver nitrate
solution (1 hr at 37°C)
Washed with water
UV Light (10 mins)
Dried and Photographed
Measured by Image Quant
Overall Fold Stimulation in Cell Count during Proliferation Stage
Control PEMF0
0.2
0.4
0.6
0.8
1
1.2
1.4
Cell Count
Fol
d C
hang
eF
old
C
han
ge
Control PEMF0.9
0.95
1
1.05
1.1
1.15
1.2
OCALPCOL1
Differentiation
Fol
d C
hang
eF
old
Ch
ange
Overall Fold Stimulation for Differentiation Stage
Control PEMF0
0.2
0.4
0.6
0.8
1
1.2
OCALPCOL 1
Mineralization
Fol
d C
hang
eF
old
C
han
geOverall Fold Stimulation for Mineralization Stage
Von Kossa Staining – Differentiation Stage
Control
PEMF Treated
Image Quant Data for von Kossa Staining- Differentiation Stage
Control PMF17
18
19
20
21
22
23
24Differentiation Stage (Series 3)
Av
era
ge
Vo
lum
e (
10
^4
)
Von Kossa Staining – Mineralization Stage
Control
PEMF Treated
Image Quant Data for von Kossa Staining- Mineralization Stage
Control PMF0
10
20
30
40
50
60
70
80
90
100
Mineralization Stage (Series 3)
Ave
rag
e V
olu
me
(10^
4)