1. 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

2.  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.  Two different pathways examined ◦ TGF-B1  Recall:

4.  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.  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.  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.  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.  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.  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.  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.

11.  Hypothesis #2 ◦ B-catenin nuclear translocation is mediated by the TGF-B signaling pathway

12.  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.  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

14.  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 CK Iε

15.  Hypothesis #3 ◦ TGF-B1 and nuclear B-catenin exert similar biological effects on MSCs

16.  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.  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.  Hypothesis #4 ◦ Nuclear B-catenin is required for primary effects of TGF-B1 on MSCs through regulation of specific downstream target genes

19.  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

20.  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

21.  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

22.  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.  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?