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TGF-?%20Signaling%20in%20Stem%20Cells%20

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Title: TGF-?%20Signaling%20in%20Stem%20Cells%20


1
TGF-? Signaling in Stem Cells Cancer
  • L Mishra, R Derynck, B Mishra
  • Science October 2005

Helen Hwang4.22.2009
2
Germ layers eventually give rise to all of an
animals tissues and organs.
  • fertilization
  • zygote
  • blastula ball
  • gastrula 3 germ layers
  • organogenesis

3
Embryonic stem cells develop into multiple
functional cell lineages.
  • ES differentiation - process where less
    specialized ? more specialized cell
  • from pluripotent ? progenitor ? functional cells
  • hematopoietic (bone marrow) ? RBC, WBC,
    platelets
  • mesenchymal (bone marrow) ? stromal, fat, bone
  • epithelial ? skin
  • neuronal
  • Cell signaling controls differentiation
  • via growth factors

ES growing on fibroblasts
4
TGF-? signaling underlies progression of
differentiation.
  • maintains undifferentiated state
  • initiates differentiation
  • specifies germ layer differentiation
  • depends on
  • stage of target cell
  • local environment
  • identity/dosage of ligand

http//stemcells.nih.gov, 2001
5
TGF-? family transforming/tumor growth factor
  • includes 30 structurally related growth factors
  • TGF-?s
  • activins
  • BMPs (bone morphogenetic protein)
  • myostatin
  • 2 types of serine-tyrosine receptors (type I
    II)
  • functionally
  • promote or inhibit cell proliferation
  • promotes apoptosis
  • differentiation

6
BMP TGF-? signaling involve SMAD proteins.
7
Differentiation of neural stem cells involves
TGF-?
  • early
  • BMPs
  • inhibits differentiation
  • after progenitors established
  • promotes differentiation (ganglions, olfactory
    neurons)
  • accelerates differentiation lineage commitment
    of precursor cells
  • fully differentiated
  • inhibits growth of normal glial cells (tumors)

8
TGF-? signaling in hematopoietic stem cells is
complex
  • early specification
  • TGF-? inhibits early multipotent hematopoietic
    stem cells (in vitro)
  • BMPs
  • promote specification
  • differentiation
  • proliferation
  • progression along lineage
  • myeloid promoted by Smad 7
  • lymphoid inhibited by Smad7
  • dependent on exogenous factors (other growth
    factors, cross-talk)

9
TGF-? Signaling in mesenchymal stem cells
  • promotes specification
  • allow mesenchymal cells from one lineage to
    switch to another lineage (pre-adipocytes ?
    osteoblasts)
  • inhibits progression and maturation of myoblasts
    (myostatin), osteoblasts (BMPs), and adipocytes
    (myostatin)
  • TGF-? expression are activated in response to
    injury (wound repair)

10
TGF-? Signaling in gastrointestinal tissues
cancer
  • tumor supressors
  • inhibits cell growth / cancer in gut epithelial
  • inactivation of any signaling ? GI tumor
  • BMP signalling
  • suppresses Wnt signaling effects ? limits cell
    renewal
  • mutations in R Smad4 ? intestinal polyposis or
    Cowden disease
  • TGF-? signaling (Smad2, Smad3, ELF) all
    necessary for proper liver and biliary system
    development
  • knockouts hepatocellular carcinoma

polyposis
hepatocellular carcinoma
11
Conclusion
  • TGF-? is a key regulator in ES differentiation
    and progression of cell lineage of progenitor
    cells
  • Environmental factors and cross-talk b/t
    pathways could affect differentiation
  • When TGF-? pathway is deregulated, depending on
    the stage ? impaired differentiation and may
    become cancerous!

12
Smad3-dependent translocation of ??-catenin is
required for TGF-b1-induced proliferation of bone
marrow-derived adult human mesenchymal stem cells
  • Hongyan Jian, Xing Shen, Irwin Liu
  • Mikhail Semenov, Xi He, Xiao-Fan WangGenes
    Development, 2006.

13
Mesenchymal stem cells (MSC) differentiate into
bone, muscle, tendon, adipose.
  • derived from bone marrow
  • TGF-? involved in wound repair

14
TGF-?1 pathway activates transcription via SMAD
proteins.
15
WNT pathway activates transcription via ?-catenin
16
Question What kind of regulatory mechanisms
underlie the renewal and differentiation of MSC?
17
TGF-??1 induces nuclear translocation of
?-catenin independently of the Wnt signaling
pathway
  • incubated MSC with Wnt3A (6h) or TGF-? medium
    (2h)
  • measured presence of ?-catenin via Western
    blotting
  • ? detected nucleus translocation for both

18
TGF-?1 induces nuclear translocation of ?-catenin
independently of the Wnt signaling pathway
with immunofluorescence imaging, nuclear staining
of endogenous ?-catenin increased in MSC 1h after
treatment with TGF-?1 Hoechst dye stains DNA
(visualize nuclei or mitochondria)
19
?-catenin nuclear translocation is associated
with certain cell types (MSCs)
  • Are TGF- ?1 effects only associated with certain
    cellular contexts?
  • take Madin-Darby canine kidney epithelial cells
    (shown), and human fibroblasts, and human
    melanocytes
  • TGF-? and Wnt3A treatment
  • TGF-?1 did not induce ?-catenin accumulation
    although Wnt3A treatment did.

20
TGF-?1s translocation activity is not mediated
by Wnt proteins.
  • Is ?-catenin translocation a consequence of
    TGF-?1 induced Wnt production action?
  • MSCs were pretreated with protein translation
    inhibitor (cyclohexmide) for 1 hr
  • treat with TGF- ?1 for 2 hrs
  • detect presence of ?-catenin,? ?-tubulin, lamin
  • ? CHX had no effect on TGF- ?1s effect

21
b-catenin is dependent on TGF-b type 1 receptor
  • Treat MSC with SD208 (kinase inhibitor of TGF-?
    type 1 receptor)
  • apply TGF-?1
  • ? Smad2 phosphorylation is blocked and ?-catenin
    translocation blocked

22
SMads are directly involved in b-catenin
translocation (via Smad-KDs)
  • introduce Smad specific siRNA (Smad3 protein
    reduced by gt90)
  • apply TGF-?1 in MSC
  • examine ?-catenin nuclear translocation
  • ? ?-catenin protein is barely detectable in
    Smad-KDs

23
TGF-?1 and nuclear ?-catenin both increase
proliferation
  • b-catenin mutants formed via retroviral infection
  • full transcriptional activity
  • alanine instead of serine p sites so unable to
    degrade (via ubiquitin)
  • treat with TGF- ?1 or untreated in control or
    mutant b-catenin/vector
  • treat with H3-thymidine
  • measure relative proliferation of human MSCs
  • ? TGF- ?1 ?-catenin mutants both have increased
    relative proliferative activity

24
TGF-B1 and nuclear b-catenin are both
anti-osteogenic
  • osteogenic assay measure alkaline phosphate
    activity
  • culture MSCs in osteogenic (OS) medium
  • treat in presence/absense of TGF-? or look at
    ?-catenin mutants
  • TGF-b and b-catenin inhibits the osteogenic
    effect of the OS medium on MSCs.
  • perhaps direct correlation between ?-catenin and
    TGF- ?1

25
TGF-b1 mediates proliferative effect on MSCs via
b-catenin translocation
  • LEF1 transcription factor that complexes with
    ?-catenin that translocates into nucleus via HMG
    box (where LEF1 Smad3 interacts)
  • LEF1?C - a mutant of LEF1 that can still complex
    with ?-catenin in cytoplasm, but cannot
    translocate into nucleus (unable to associate
    with Smad3)
  • In LEF1?C, TGF-?1 did not induce proliferation.
  • In LEF1?C, TGF-?1 did not inhibit osteogenic
    differentiation.
  • ? ?-catenin is required for TGF-? to exert some
    of its biological effects on MSCs.

26
Some unanswered questions
  • Interactions exist in other cells, but why does
    SMAD3 only work in MSCs?
  • Opposite physiological effects seen in human
    MSCs as opposed to other stem-cell types. Why?
  • How do TCF/LEF transcription factors participate
    in the proliferative response seen?
  • What kind of downstream mechanisms exist after
    SMAD3 but before ?-catenin in the Wnt and TGF-?
    pathway?

27
Conclusions
  • Smad3 plays a role in the translocation of
    b-catenin into nucleus through a process
    initiated by TGF-b1
  • This is a novel signaling pathway found only in
    MSCs
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