Differentiation of neural stem cells involves TGF- early: – BMPs inhibits differentiation after progenitors established: – promotes differentiation (ganglions, olfactory neurons) – accelerates differentiation & lineage commitment of precursor cells fully differentiated: – inhibits growth of normal glial cells (tumors)
TGF- signaling in hematopoietic stem cells is complex early specification: – TGF- inhibits early multipotent hematopoietic stem cells (in vitro) – BMPs promote specification differentiation proliferation progression along lineage – myeloid: promoted by Smad 7 – lymphoid: inhibited by Smad7 –dependent on exogenous factors (other growth factors, cross-talk)
TGF- Signaling in mesenchymal stem cells promotes specification –allow mesenchymal cells from one lineage to switch to another lineage (pre-adipocytes osteoblasts) inhibits progression and maturation of myoblasts (myostatin), osteoblasts (BMPs), and adipocytes (myostatin) TGF- expression are activated in response to injury (wound repair)
TGF- Signaling in gastrointestinal tissues & cancer tumor supressors – inhibits cell growth / cancer in gut epithelial –inactivation of any signaling GI tumor BMP signalling –suppresses Wnt signaling effects limits cell renewal –mutations in R & Smad4 intestinal polyposis or Cowden disease TGF- signaling (Smad2, Smad3, ELF) all necessary for proper liver and biliary system development – knockouts – hepatocellular carcinoma hepatocellular carcinoma polyposis
Conclusion – TGF- is a key regulator in ES differentiation and progression of cell lineage of progenitor cells – Environmental factors and cross-talk b/t pathways could affect differentiation – When TGF- pathway is deregulated, depending on the stage impaired differentiation and may become cancerous!
Smad3-dependent translocation of -catenin is required for TGF-b1-induced proliferation of bone marrow-derived adult human mesenchymal stem cells Hongyan Jian, Xing Shen, Irwin Liu Mikhail Semenov, Xi He, Xiao-Fan Wang Genes & Development, 2006.
Mesenchymal stem cells (MSC) differentiate into bone, muscle, tendon, & adipose. derived from bone marrow TGF- involved in wound repair
TGF- 1 pathway activates transcription via SMAD proteins.
WNT pathway activates transcription via -catenin
Question: What kind of regulatory mechanisms underlie the renewal and differentiation of MSC?
TGF- 1 induces nuclear translocation of -catenin independently of the Wnt signaling pathway incubated MSC with Wnt3A (6h) or TGF- medium (2h) measured presence of -catenin via Western blotting detected nucleus translocation for both
TGF- 1 induces nuclear translocation of -catenin independently of the Wnt signaling pathway with immunofluorescence imaging, nuclear staining of endogenous - catenin increased in MSC 1h after treatment with TGF- 1 Hoechst dye – stains DNA (visualize nuclei or mitochondria)
-catenin nuclear translocation is associated with certain cell types (MSCs) Are TGF- 1 effects only associated with certain cellular contexts? take Madin-Darby canine kidney epithelial cells (shown), and human fibroblasts, and human melanocytes TGF- and Wnt3A treatment TGF- 1 did not induce -catenin accumulation although Wnt3A treatment did.
TGF- 1’s translocation activity is not mediated by Wnt proteins. Is -catenin translocation a consequence of TGF- 1 induced Wnt production & action? MSCs were pretreated with protein translation inhibitor (cyclohexmide) for 1 hr treat with TGF- 1 for 2 hrs detect presence of -catenin, -tubulin, lamin CHX had no effect on TGF- 1’s effect
b-catenin is dependent on TGF-b type 1 receptor Treat MSC with SD208 (kinase inhibitor of TGF- type 1 receptor) apply TGF- 1 Smad2 phosphorylation is blocked and -catenin translocation blocked
SMads are directly involved in b-catenin translocation (via Smad-KDs) introduce Smad specific siRNA (Smad3 protein reduced by >90%) apply TGF- 1 in MSC examine -catenin nuclear translocation -catenin protein is barely detectable in Smad-KDs
TGF- 1 and nuclear -catenin both increase proliferation - treat with TGF- 1 or untreated in control or mutant b- catenin/vector - treat with H3-thymidine - measure relative proliferation of human MSCs TGF- 1 & -catenin mutants both have increased relative proliferative activity -b-catenin mutants formed via retroviral infection -full transcriptional activity -alanine instead of serine p sites so unable to degrade (via ubiquitin)
TGF-B1 and nuclear b-catenin are both anti-osteogenic osteogenic assay – measure alkaline phosphate activity -culture MSC’s in osteogenic (OS) medium -treat in presence/absense of TGF- or look at -catenin mutants TGF-b and b-catenin inhibits the osteogenic effect of the OS medium on MSCs. perhaps direct correlation between -catenin and TGF- 1
TGF-b1 mediates proliferative effect on MSCs via b-catenin translocation -LEF1 – transcription factor that complexes with -catenin that translocates into nucleus via HMG box (where LEF1 & Smad3 interacts) -LEF1ΔC - a mutant of LEF1 that can still complex with -catenin in cytoplasm, but cannot translocate into nucleus (unable to associate with Smad3) -In LEF1ΔC, TGF- 1 did not induce proliferation. -In LEF1ΔC, TGF- 1 did not inhibit osteogenic differentiation. -catenin is required for TGF- to exert some of its biological effects on MSCs.
Some unanswered questions – Interactions exist in other cells, but why does SMAD3 only work in MSCs? – Opposite physiological effects seen in human MSCs as opposed to other stem-cell types. Why? – How do TCF/LEF transcription factors participate in the proliferative response seen? – What kind of downstream mechanisms exist after SMAD3 but before -catenin in the Wnt and TGF- pathway?
Conclusions Smad3 plays a role in the translocation of b-catenin into nucleus through a process initiated by TGF-b1 This is a novel signaling pathway found only in MSCs