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David Mahr Graduate Student

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David Mahr Graduate Student

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  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-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. 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/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

  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. • 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

  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. • 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. 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 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. 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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