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)