Smad3-dependent nuclear translocation of B-catenin is required for TGF-B1-induced proliferation of bone marrow-derivced adult human mesenchymal stem cells

Hongyan Jian1, Xing Shen1, Irwin Liu1, Mikhail Semenov2, Xi He2, Xiao-Fan Wang1

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-

catenin into the nucleus?