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David Mahr
Graduate Student

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
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Method: Identify key regulators in mechanisms and pathways
via “knock-out” methods

Two different pathways examined
◦ TGF-B1
 Recall:

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

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

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

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
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

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

Examine whether TGF-B1 induced B-catenin NT requires Wnt
signaling (same question)
◦ MSCs pretreated with Wnt signal disruptor, DVL-ΔPDZ, before addition of Wnt and
TGF-B1
• DVL-ΔPDZ did not have an effect on TGF-B1 induced B-catenin NT
• DVL-ΔPDZ inhibited Wnt3A induced B-catenin NT (not shown)
• TGF-B1 induced B-catenin NT does not require the canonical Wnt signaling pathway.

Hypothesis #2
◦ B-catenin nuclear translocation is mediated by the TGF-B
signaling pathway

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

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
Cytosol
Nucleus
•
•
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

•
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
•
•
Smad3/GSK-3B complexes identified
•
•
Association decreases with addition of TGF-B1
Smad3/Axin/CKIε existence known from previous work
•
•
Association uneffected by addition of TGF-B1
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 CKIε

Hypothesis #3
◦ TGF-B1 and nuclear B-catenin exert similar biological effects
on MSCs

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

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-cateninmediated TGF-B1 signaling pathway
and its unique biological responses in
MSCs
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Hypothesis #4
◦ Nuclear B-catenin is required for primary effects of TGF-B1 on
MSCs through regulation of specific downstream target genes

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.
◦ LEF1ΔC, 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 LEF1ΔC
TGF-B1 induced cell profileration inhibited of LEF1Δ
TGF-B1 induced osteogenic differentation inhibited of LEF1Δ
•
Supports that B-catenin NT is required for TGF-B1 to exert its biological effects on MSCs

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.
◦ LEF1ΔC, Mutant LEF: Unable to form complex with B-catenin or interact with Smad3
•
•
•
TGF-B1 unable to induce B-catenin NT in presence of LEF1ΔC
TGF-B1 induced cell profileration inhibited in presence of LEF1ΔC
TGF-B1 inhibition of osteogenic differentation inhibited in presence of LEF1ΔC
•
Supports that B-catenin NT is required for TGF-B1 to exert its biological effects on MSCs

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 LEF1ΔC present.
BAX induced by TGF-B1 signaling with both LEF1 and LEF1ΔC 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
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Demonstrates existence TGF-B1 induced B-catenin nuclear
translocation pathway mediated by Smad3
◦ Signaling pathway specific to MSCs
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TGF-B1 exerts biological effects on MSCs
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Overlap and cross-talk of different pathways/protiens yields end
biological effects
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Future Research: To further understanding of these
mechanisms and enable the ability to control cell proliferation
and differentiation
◦ Proliferation of MSCs
◦ Inhibition of osteogenic differentiation

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
Bcatenin into the nucleus?