Title: TGF-?%20Signaling%20in%20Stem%20Cells%20
1TGF-? Signaling in Stem Cells Cancer
- L Mishra, R Derynck, B Mishra
- Science October 2005
Helen Hwang4.22.2009
2Germ layers eventually give rise to all of an
animals tissues and organs.
- fertilization
- zygote
- blastula ball
- gastrula 3 germ layers
- organogenesis
3Embryonic 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
4TGF-? 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
5TGF-? 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
6BMP TGF-? signaling involve SMAD proteins.
7Differentiation 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)
8TGF-? 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)
9TGF-? 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)
10TGF-? 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
11Conclusion
- 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!
12Smad3-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.
13Mesenchymal stem cells (MSC) differentiate into
bone, muscle, tendon, adipose.
- derived from bone marrow
- TGF-? involved in wound repair
14TGF-?1 pathway activates transcription via SMAD
proteins.
15WNT pathway activates transcription via ?-catenin
16Question What kind of regulatory mechanisms
underlie the renewal and differentiation of MSC?
17TGF-??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
18TGF-?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.
20TGF-?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
21b-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
22SMads 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
23TGF-?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
24TGF-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
25TGF-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.
26Some 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?
27Conclusions
- 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