Transcript Slide 1
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 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 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 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 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 ◦ 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?