TGF-  Signaling in Stem Cells & Cancer

1. TGF- Signaling in Stem Cells & Cancer Helen Hwang4.22.2009 L Mishra, R Derynck, B Mishra Science: October 2005

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

3. 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

4. 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 http://stemcells.nih.gov, 2001

5. 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

6. BMP & TGF- signaling involve SMAD proteins.

7. 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) Differentiation of neural stem cells involves TGF-

8. 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 hematopoietic stem cells is complex

9. 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)

10. 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 polyposis hepatocellular carcinoma

11. 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!

12. 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 WangGenes & Development, 2006.

13. Mesenchymal stem cells (MSC) differentiate into bone, muscle, tendon, & adipose. derived from bone marrow TGF- involved in wound repair

14. TGF-1 pathway activates transcription via SMAD proteins.

15. WNT pathway activates transcription via -catenin

16. Question: What kind of regulatory mechanisms underlie the renewal and differentiation of MSC?

17. 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

18. 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)

19. -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.

20. 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

21. 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

22. 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

23. TGF-1 and nuclear -catenin both increase proliferation • b-catenin mutants formed via retroviral infection • full transcriptional activity • alanine instead of serine p sites so unable to degrade (via ubiquitin) • 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

24. 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

25. 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.

26. 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?

27. 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