Stem Cells and Cell Signaling Cheng-En Lai BIOE 506: Molecular and Cellular Bioengineering 4/25/2011

Overview Background Transforming Growth Factor-β (TGF-β) Stem Cells Signaling Pathways Transforming Growth Factor-β (TGF-β) SMAD Signaling Pathway Stem Cell Differentiation Overview Signaling Examples TGF-β in various cell types Other Signaling Pathways WNT Notch Hedgehog Fibroblast Growth Factor (FGF) Cross-talk SMAD/WNT WNT/FGF/Notch/SMAD/Hedgehog Conclusions

Background: Stem Cells Self Renewal Pluripotency Source for tissue engineering and cell replacement therapies Similar to cancer cells; stem cells thought to be derived from cancer stem cells Adapted from System Biosciences (systembio.com) Understanding stem cells is important for understanding cancer

Background: Signaling Pathways Abnormalities in pathways may give rise to cancer stem cells and tumors Understanding the signaling pathways and identifying important factors helps to understand cancer transformation as well stem cell differentiation for tissue engineering and regenerative medicine applications

Transforming Growth Factor β TGF-β proteins and TGF-β related bone morphogenetic proteins (BMPs) are important regulators of stem cell differentiation, maintenance, and self-renewal, as well as carcinogenesis suppression. Comprised of 30 related proteins in the SMAD pathway

SMAD Signaling Pathway The downstream canonical signaling pathway for TGF-β proteins, including activin, nodal, and BMP. Also called the nodal signaling pathway and TGF-beta signaling pathway. Bind to type 1/type II serine/theonine kinase receptors. A number of SMAD proteins are activated via phosphorylation Receptor SMADs complex with common-partner SMAD4 to translocate to the nucleus Inhibitory SMADs inactivate R-Smads and function as a negative feedback loop. Signaling cascade resulting in activation and suppression of genes, such as OCT3/4 and Nanog. OCT3 OCT4 Nanog Adapted from Blank et al. (2008)

Stem Cell Differentiation Overview BMP-4 is important for proliferation and is mediated by its inhibitory effect on neural differentiation Shows how changes in signal levels drive differentiation. IE. BMP signals alone are required for formation of the posterior primitive streak, and eventually giving rise to mesodermal tissue M=maintenance D=differentiation Adapted from Watabe et al. (2009)

SMAD Signaling Examples Nodal and activin cooperate with the WNT pathway to maintain ES cells and keep them undifferentiated and pluripotent. Activin and TGF-β confers mesodermal differentiation depending on amount. BMP signaling results in mesodermal and ectodermal differentiation in human ES cells. Nodal signals are important for OCT3/4 expression and maintenance of ES cells. Activin is important for maintenance of pluripotency, which is possibly done through induction of Nanog and OCT-4 OCT4 and Nanog are important transcription factors in generation of IPS cells BMP and leukemia-inhibiting factor keep mouse ES cells in the undifferentiated state.[Mishra]

TGF-β in Neural Stem Cells BMP inhibits neural differentiation TGF-β promotes differentiation in committed progenitors Inactivation of TGF-β growth-inhibitory functions result in tumor progression Adapted from Mishra et al. (2005).

TGF-β in other Cell Types Hematopoietic Stem cells Inhibits early progenitors, while enhances differentiation of committed stem cells Mesenchymal Stem Cells Inhibits differentiation and maturation into myoblasts, osteoblasts, and adipocytes, while stimulating MSC proliferation Basis for efficient wound repair in mesenchymal tissue Gastrointestinal Epithelial Stem Cells Inactivation with one TGF-β component (Receptor, SMAD protein) is present in all gastrointestinal cancer BMP’s and cytokines promote specification, differentiation and proliferation of human ES cells Deregulation of TGF-β can result in cancer progression as well. BMPs activate osteoblast differentiation.

WNT Signaling Pathway Adapted from Katoh et al. (2007). Frizzled receptors and LRP coreceptors receive canonical WNT signals, which in turn activate disheveled proteins (DVL). DVL proteins inhibit the formation of the axin/GSK-3/APC complex, which acts to phosphorylate beta-catenin, tagging it for polyubiquitination and eventual degradation. Nuclear accumulation of beta-catenin complexes with TCF/LEF transcription factors which activate FGF20, DKK1, WISP1, MYC, and CCND1 genes. Adapted from Katoh et al. (2007).

WNT Signaling Pathway Cell fate determination Transformation of cancer stem cells due to disregulation MYC is a transcription factor involved in generation of IPS cells.

Notch Signaling Pathway Delta or jagged ligands bind to Notch receptors. Gamma-secretase causes the release of the intracellular domain (NICD) Forms a complex with CSL and MAM and activate genes involved in neural differentiation. Adapted from Bray et al. (2009).

Notch Signaling Pathway Promotion of neural cell differentiation Involved in self-renewal of hematopoietic stem cells

Hedgehog Signaling Pathway Shh binds to Patched-1 which stops inhibition of Smoothened. Smo activates Gli proteins in the nucleus resulting in regulation of genes. Adapted from Altaba et al. (2002).

Hedgehog Signaling Pathway Induces differentiation of hematopoietic progenitors and neural stem cells Skin, muscle, and brain cancers develop when pathway is maintained improperly in stem cells

FGF Signaling Pathway Adapted from Katoh et al. (2006) FGF signals are transduced through FGF receptors Various signaling cascades result in EMT, cell survival, and proliferation/differentiation EMT is caused by downregulation of E-cadherin Adapted from Katoh et al. (2006)

FGF Signaling Pathway EMT Cell survival Proliferation/differentiation Cross-talk is seen between WNT and FGF via down-regulation of GSK3β, resulting in tumors with more malignant phenotypes of mammary carcinogenesis

TGF-β1 /WNT Pathway Cross-talk SMAD and TCF/LEF associate to cooperatively regulate genes Series of experiment by Jian et. al. (2006) show that TGF-β1 addition results in rapid nuclear accumulation of β-catenin in MSCs in a new form of cross-talk. β-catenin nuclear accumulation is not due to phosphorylation as from canonical WNT pathway Mediated by SMAD3/GSK3β disruption through TGF-β mediated phosphorylation. MSCs act differently with TGF-β. SMAD3 acts as a chaperone for beta-catenin

TGF-β1 /WNT Pathway Cross-talk MSCs act differently with TGF-β. SMAD3 acts as a chaperone for beta-catenin

WNT/FGF/Notch/SMAD/Hedgehog Cross-talk Balance of all signaling pathways is important for homeostasis and prevention of cancer and congenital diseases Hedgehog pathway SMAD pathway Notch family receptor Notch family receptor Hedgehog pathway induced

Conclusions Many signaling pathways with cross talk involved in stem cell proliferation, maintenance, and differentiation Dependent on differentiation stage, type of cell, local environment, and the identity and amount of particular ligand Identification of key regulators has potential for generation of iPS cells and cell replacement therapies

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