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 - PowerPoint PPT Presentation

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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

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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


1
Smad3-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

2
Introduction
  • 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

3
Introduction
  • Two different pathways examined
  • TGF-B1
  • Recall

4
Introduction
  • 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
5
Results
  • 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

6
Results - 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

7
Results - 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

8
Results - 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

9
Results - 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

10
Results - 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.

11
Results
  • Hypothesis 2
  • B-catenin nuclear translocation is mediated by
    the TGF-B signaling pathway

12
Results - 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

13
Results - 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

14
Results - 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

15
Results
  • Hypothesis 3
  • TGF-B1 and nuclear B-catenin exert similar
    biological effects on MSCs

16
Results 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

17
Results - 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

18
Results
  • Hypothesis 4
  • Nuclear B-catenin is required for primary effects
    of TGF-B1 on MSCs through regulation of specific
    downstream target genes

19
Results - 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

20
Results - 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

21
Results - 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

22
Conclusion
  • 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

23
Critiques
  • 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?

24
Questions?
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