Smad3-dependent nuclear translocation of B-catenin is required for TGF-B1-induced proliferation of bone marrow-derivced adult human mesenchymal stem cells Hongyan Jian 1 , Xing Shen 1 , Irwin Liu 1 , Mikhail Semenov 2 , Xi He 2 , Xiao-Fan Wang 1 1 Department of Pharmacology and Cancer Biology, Duke University Medical Center 2 Division of Neuroscience, Childrens Hospital and Harvard Medical School David Mahr Graduate Student
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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
1Department of Pharmacology and Cancer Biology, Duke University Medical Center2Division of Neuroscience, Childrens Hospital and Harvard Medical School
David MahrGraduate 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
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
• 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
Cytosol Nucleus
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/CKIε 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 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-catenin-mediated TGF-B1 signaling pathway and its unique biological responses in MSCs
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
• 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
B-catenin Levels
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
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
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-