Title: Smad3-dependent nuclear translocation of B-catenin is required for TGF-B1-induced proliferation of bone marrow-derivced adult human mesenchymal stem cells Hongyan Jian1, Xing Shen1, Irwin Liu1, Mikhail Semenov2, Xi He2, Xiao-Fan Wang1 1Department of
1Smad3-dependent nuclear translocation of
B-catenin is required for TGF-B1-induced
proliferation of bone marrow-derivced adult human
mesenchymal stem cellsHongyan Jian1, Xing
Shen1, Irwin Liu1, Mikhail Semenov2, Xi He2,
Xiao-Fan Wang11Department of Pharmacology and
Cancer Biology, Duke University Medical
Center2Division of Neuroscience, Childrens
Hospital and Harvard Medical School
- David Mahr
- Graduate Student
2Introduction
- Adult Mesenchymal Stem Cells
- Source of regenerative mesenchymal tissue
- Differentiate into bone, cartilage, muscle,
tendon, and adipose. - Goal To understand mechanisms of proliferation
and differentiation - Method Identify key regulators in mechanisms and
pathways via knock-out methods
3Introduction
- Two different pathways examined
- TGF-B1
- Recall
4Introduction
- Two different pathways examined
- Wnt Pathway
- Recall
Wnt ligand binds FRZ receptor
Activates DSH protein
DSH inactivates axin/GSK/APC
Increases B-catenin level
B-catenin gene expression
5Results
- Hypothesis 1
- TGF-B1 induces nuclear translocation of B-catenin
without affecting the steady-state protein level
of B-catenin and is independent of the Wnt
signaling pathway
6Results - Hypothesis 1
- Examine whether TGF-B1 induces B-catenin nuclear
translocation - MSCs stimulated with Wnt3A and TGF-B1
- Stained with B-catenin specific antibody
- TGF-B1 induced nuclear translocation of
B-catenin in MSCs
7Results - Hypothesis 1
- Examine whether TGF-B1 effects are cell specific
- MDCK cells treated with TGF-B1 and Wnt3A
- Nuclear B-catenin levels in MDCK cells did not
increase in response to TGF-B1 - TGF-B1 induced B-catenin nuclear translocation
may be associated specifically with MSCs
8Results - Hypothesis 1
- Examine whether TGF-B1 induced B-catenin NT
requires Wnt signaling - MSCs pretreated with protein translation
inhibitor CHX before addition of TGF-B1 - Blocks autocrine mechanism of Wnt
- Presence of CHX did not have an effect on TGF-B1
induced B-catenin NT - TGF-B1 induced B-catenin NT is not mediated by
increase in production of Wnt proteins
9Results - Hypothesis 1
- Examine whether TGF-B1 induced B-catenin NT
requires Wnt signaling (same question) - MSCs pretreated with competitive inhibitor of Wnt
receptor FRZ, Fz8CRD, before addition of TGF-B1
and Wnt
- Fz8CRD did not have an effect on TGF-B1 induced
B-catenin NT - Fz8CRD inhibited Wnt3A induced B-catenin NT
(results not shown) - TGF-B1 induced B-catenin NT is not a Wnt
ligand-dependent process
10Results - Hypothesis 1
- Examine whether TGF-B1 induced B-catenin NT
requires Wnt signaling (same question) - MSCs pretreated with Wnt signal disruptor,
DVL-?PDZ, before addition of Wnt and TGF-B1
- DVL-?PDZ did not have an effect on TGF-B1
induced B-catenin NT - DVL-?PDZ inhibited Wnt3A induced B-catenin NT
(not shown) - TGF-B1 induced B-catenin NT does not require
the canonical Wnt signaling pathway.
11Results
- Hypothesis 2
- B-catenin nuclear translocation is mediated by
the TGF-B signaling pathway
12Results - Hypothesis 2
- Examine whether TGF-B1 induced B-catenin NT is
dependent on TGF-B type I receptor - MSCs pretreated with inhibitor of TGF-B type I
receptor kinase, SD208, before addition of TGF-B1
- SD208 blocked phosphorylation of Smad2 and
inhibited B-catenin NT. - TGF-B1 induced B-catenin NT is mediated by the
TGF-B signaling pathway via the type I receptor
kinase
13Results - Hypothesis 2
- Examine the effect of Smads in process of
B-catenin NT - MSCs pretreated with Smad3-siRNA to knockdown
Smad3 expression before addition of TGF-B1 - Positive control Empty retrovirus
Nucleus
Cytosol
- Lack of Smad3 expression inhibited B-catenin NT
- Wnt induced B-catenin NT present
- Smad3 required for TGF-B1 induced B-catenin NT
(Smad2 may not be involved) - Wnt3A induced B-catenin NT distinct from TGF-B1
induced B-catenin NT
14Results - Hypothesis 2
- Examine the possibility of Smad3 active transport
of B-catenin - MSCs coimmunoprecipitated with Smad3 antibody
for Smad3/B-catenin and Smad3/GSK-3B complexes
before addition of TGF-B1
- Smad3/B-catenin complexes identified
- Association uneffected by addition of TGF-B1
- Smad3/GSK-3B complexes identified
- Association decreases with addition of TGF-B1
- Smad3/Axin/CKIe existence known from previous
work - Association decreases with addition of TGF-B1
- Supports model that TGF-B1 induced B-catenin NT
can be directly linked to dynamics of a protein
complex possibly containing B-catenin, Smad3,
GSK-3B, Axin, and CKIe
15Results
- Hypothesis 3
- TGF-B1 and nuclear B-catenin exert similar
biological effects on MSCs
16Results Hypothesis 3
- Examine effects of TGF-B1 on regulation of
proliferation and osteogenic differentiation in
MSCs - Proliferation measured in presence and absence of
TGF-B1 - Osteogenic assay performed to measure ALP
production in presence and absence of TGF-B1 - MSCs cultured in osteogenic supplemental medium
(OS)
- TGF-B1 simulates proliferation of MSCs
- ALP levels reduced in presence of TGF-B1
- TGF-B1 inhibits osteogenic differentiation
17Results - Hypothesis 3
- Examine link of B-catenin NT to TGF-B1 regulation
of proliferation and osteogenic differentiation - Mutant B-catenin introduced into MSCs
- Prevents ubiquitination-mediated degradation
- Readily translocated across nucleus
- Retains transcriptional ability
- Mutant B-catenin translocated into nucleus (w/out
need of TGF-B1) - Mutant B-catenin induced profileration of MSCs
and inhibited osteogenic differentiation - Supports direct correlation between activation of
Smad3/B-catenin-mediated TGF-B1 signaling pathway
and its unique biological responses in MSCs
18Results
- Hypothesis 4
- Nuclear B-catenin is required for primary effects
of TGF-B1 on MSCs through regulation of specific
downstream target genes
19Results - Hypothesis 4
- Examine how B-catenin is required for TGF-B1
induced biological effects on MSC - LEF1 Transcription factor that forms complex
with B-catenin via N-terminal region and also
mediates Smad3 towards transcription. - LEF1?C, Mutant LEF Unable to form complex with
B-catenin or interact with Smad3
B-catenin Levels
- TGF-B1 unable to induce B-catenin NT in presence
of LEF1?C - TGF-B1 induced cell profileration inhibited of
LEF1? - TGF-B1 induced osteogenic differentation
inhibited of LEF1? - Supports that B-catenin NT is required for TGF-B1
to exert its biological effects on MSCs
20Results - Hypothesis 4
- Examine how B-catenin is required for TGF-B1
induced biological effects on MSC - LEF1 Transcription factor that forms complex
with B-catenin via N-terminal region and mediate
Smad3 towards transcription. - LEF1?C, Mutant LEF Unable to form complex with
B-catenin or interact with Smad3
- TGF-B1 unable to induce B-catenin NT in presence
of LEF1?C - TGF-B1 induced cell profileration inhibited in
presence of LEF1?C - TGF-B1 inhibition of osteogenic differentation
inhibited in presence of LEF1?C - Supports that B-catenin NT is required for TGF-B1
to exert its biological effects on MSCs
21Results - Hypothesis 4
- Examine regulation of gene expression by
B-catenin mediated TGF-B signaling pathways - Microarray analysis performed to identify TGF-B1
regulated target genes that depend on nuclear
B-catenin
- BLK induced by TGF-B1 signaling with LEF1
present, blocked with LEF1?C present. - BAX induced by TGF-B1 signaling with both LEF1
and LEF1?C present. - Nuclear B-catenin required for TGF-B1 mediated
expression of BLK - TGF-B1 mediated expression of BAX not dependent
on B-catenin - Controlled by another TGF-B pathway
22Conclusion
- Demonstrates existence TGF-B1 induced B-catenin
nuclear translocation pathway mediated by Smad3 - Signaling pathway specific to MSCs
- TGF-B1 exerts biological effects on MSCs
- Proliferation of MSCs
- Inhibition of osteogenic differentiation
- Overlap and cross-talk of different
pathways/protiens yields end biological effects - Future Research To further understanding of
these mechanisms and enable the ability to
control cell proliferation and differentiation
23Critiques
- TGF-B1 promotes proliferation in MSCs
- However, TGF-B inhibits proliferation in nearly
all other progenitor cells (Why?) - Key to understanding pathway across all cell
types - Mutant B-catenin almost completely localized in
nucleus - Previous studies have shown same mutant B-catenin
localized at the plasma membrane - What mechanisms are involved to translocate
mutant B-catenin into the nucleus?
24Questions?