TGF-  Signaling in Stem Cells & Cancer L Mishra, R Derynck, B Mishra Science: October 2005 Helen Hwang

Germ layers eventually give rise to all of an animal’s tissues and organs. fertilization zygote blastula – ball gastrula – 3 germ layers organogenesis

Embryonic stem cells develop into multiple functional cell lineages. ES differentiation - process where less specialized  more specialized cell from pluripotent  progenitor  functional cells – hematopoietic (bone marrow)  RBC, WBC, platelets – mesenchymal (bone marrow)  stromal, fat, bone – epithelial  skin – neuronal Cell signaling controls differentiation – via growth factors ES growing on fibroblasts

TGF-  signaling underlies progression of differentiation. maintains undifferentiated state initiates differentiation specifies germ layer differentiation depends on: –stage of target cell –local environment –identity/dosage of ligand

TGF-  family “transforming/tumor growth factor” includes 30 structurally related growth factors – TGF-  s – activins – BMPs (bone morphogenetic protein) – myostatin 2 types of serine-tyrosine receptors (type I & II) functionally: promote or inhibit cell proliferation promotes apoptosis differentiation

BMP & TGF-  signaling involve SMAD proteins.

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