LECTURE COURSE „ Developmental Biology“ Introduction to Developmental Biology

1. LECTURE COURSE „Developmental Biology“ • Introduction to Developmental Biology • II. Basic Principles and Mechanisms of Developmental Biology (Gastrulation and Organogenesis) • III. Stem Cells • IV. Growth Factors and Signal Transduction

2. Definition I:growth factors are small proteins, present in animal tissues at very low concentrations, but having enormously high biological activity* that are responsible for controlling some of the most essential of biological functions of cells, such as growth, differentiation, migration, and survival Growth factors show several specific features: • Growth factors vs. cytokines, cytokinines • Growth factors vs. hormones • Growth factors vs. mechanism of active secretion and/or passive • release • Growth factors vs. extracellular matrix • Growth factors vs.cancer • Growth factors vs. specific receptors, signalling pathways and • signal transduction * ~10-9 – 10-11 M

3. Definition II:signalling pathways are activated by complex mechanisms where growth factors first exert their action by binding to specific receptors on the surface of the target cells. These receptors then produce metabolic changes within the target cell, which eventually activate or repress specific genes to bring about a change in cellular behaviour Intracellular signal transduction is highly specific for each growth factor and its cognate receptor. However, signal transduction can be modified at several levels and signal transduction pathways triggered by different growth factors can intensively interact to enhance or inhibit resulting cell response !!! Major characteristics of signal transduction pathways: • The ability of cells to accept signal and initiate the mechanism of signal transduction that eventually results • in cell response is defined by the presence or absence of specific receptors on the surface of cells • The presence or absence of membrane receptors is controlled by genetic programme that is specific • for each cell type • The number of receptors present on the surface of the target cells is critical for subsequent signal • transduction • Cell response involves dramatic changes in gene expression, changes in cytoskeleton and changes • in metabolism of cells • Any deregulation of the activity of growth factors and their receptors at any level of signal • transduction may cause serious defects !!!

4. The history of growth factors, growth factor receptors and signal transduction 1940 – 1960 Nerve gowth factor (NGF) -Rita Levi-Montalcini and Stanley Cohen ? Small proteins that are produced by limited group of cells cause sprouting and elongation of sensory and sympathetic neurons 1962 – 1980 Epidermal growth factor (EGF) and its receptor (EGFR) -Stanley Cohen Small proteins bind to their specific receptors and cause phosphorylation of the cell membrane and several downstream proteins to eventually activate gene transcription EGF EGF P P P P = signal transduction P … Amgen & Epo & G-CSF

5. Signal transduction is the process by which cells communicate with their environment and respond temporally to external cues (e.g. growth factors) that they sense there • The membrane receptors are inside of cells linked to • Ion channels • G proteins • Enzymes/Protein kinases The nature of interplay between membrane receptors and downstream signalling cascade defines cell response !!! Ion channels-allow the flow of ions down their electrochemical gradients G proteins-allow the membrane trafficking and activation (formation of complexes) of membrane enzymes with signalling activities Receptor kinases-allow the change in enzymatic properties and directly activate downstream signalling cascade

6. Function and three-dimensional structure of ion channels ~106 ions per second Ion channel proteins act as gates that span the lipid bilayer that surrounds all cells where they open and close to allow the flow of ions depending on their electrochemical gradients. Their central role is in the function of the EXCITABLE tissues (heart, brain, muscles, nervous system) Ion chnnels generate and propagate the electrical signals that allow us to sense our surroundings, process information, make decision, and move !!! EXAMPLE:chemical signal in the form of neural mediator acetylcholine causes structural changes in conformation of ion channel and ion channels open and close to allow the flow of ions (Na+,K+,Ca2+…). This results in establishing the electrochemical gradient that thereafter causes further changes in transmembrane voltage and changes in multimeric assembly of ion channel protein subunits This complex mechanism leads to NERVE IMPULSE

7. G protein organization and signalling Receptors linked to G proteins have common structure 1. G proteins are composed of three subunits - , , and  2. …and mediate cell response to large number of extracellular stimuli, including polypeptides, hormones, neurotransmitters, photons, odorants, and even ions GDP – guanosindiphosphate GTP - guanosintriphosphate Mode of action: Receptor (R) is activated by specific ligand (A) and this initiates the conformational changes that cause receptor activation (R). R then associates with G-GDP, G/G and mediate release of GDP from G subunit and its change to GTP. The binding of GTP to G dissociates G/G from activated receptor, and all subunits of G protein are then able to activate downstream effectors. The GTP hydrolysis to GDP terminates the action. The turn-off reaction can be accelerated either by effector itself or by proteins termed regulators of G protein signalling (RGSs) 1. 2.

8. Protein kinase receptor overview Extracellular domains ligand transmembrane domain cytoplazm Catalytic domains Receptor dimerization • Structure of receptor kinases: • Extracellular ligand binding domains • Short transmembrane domain • Intracellular kinase domains The most important family of protein kinase receptors are protein tyrosine kinase receptors that transduce signals regulating cell growth, differentiation, survival, and migration Mode of action:protein tyrosine kinase receptors are activated by ligand-induced dimerization. This brings the receptor kinase domains close to each other, which results in autophosphorylation within the intracellular kinase domains. The autophosphorylation occurs on tyrosine residues and triggers downstream signalling cascade that consists of recruiting and activation of multiple signal molecules

9. Functionally different group of receptors represent NUCLEAR RECEPTORS Nuclear receptors comprise a group of intracellular proteins found either in the cytoplasm, weakly bound in the nucleus, or both, that, in combination with small molecule ligands, bind tightly in the nucleus, recruit appropriate co-factors, and regulate transcription of target genes • Included in this family of receptors are • receptors for the steroid hormones • receptors for thyroid hormone • receptors for retinoids

10. Protein tyrosine kinase (PTK) receptor family Fibroblast growth factor receptors (FGFR) • In the human genome, 58 genes encode PTK receptors • Based on their overall structures, they can be placed into 20 subfamilies • Overactivity of PTK receptors has been implicated in number of diseases, particularly cancer. Several of PTK receptors were identified as transforming oncogene products !!!

11. Example: Fibroblast growth factors (FGFs) and their receptors • large family of signalling polypeptides (22 members by 2006) • expressed in various cell types from early embryos to adults • range in 16 to 34 kDa (molecular mass isoforms) • highly conserved across vertebrates • interact with extracellular matrix and membrane-bound heparan sulfate proteoglycans and their fragments (low-affinity receptors) • bind to four high-affinity PTK receptors that are activated upon dimerization • function in multiple biological processes including proliferation, differentiation, migration, response to injury, tissue repair, malignant transformation, regulation of cell survival and apoptosis, neovascularization … Fibroblast growth factors function as morphogens !!! Fibroblast growth factor receptors are oncogenic kinases !!!

12. „Canonical“ signalling pathway of FGFs and FGFRs Membrane proteoglycans Cell membrane „docking site“ SH2 domain containing protein Ras/Raf/MEK/ERK signalling pathway is crucial in cancer !!! • Genetic abnormalities found • in FGFRs that participate in cancer: • Chromosomal translocations • Gene amplification and overexpression • Activating mutations Changes in gene expression and cell response

13. „Canonical“ signalling pathway of FGFs and FGFRs Membrane proteoglycans Cell membrane „docking site“ IMPORTANT: Depending on complex regulation of the signal transduction and huge differences in phenotype of cells, the effects of growth factors can be very different in different cell types !!! SH2 domain containing protein Ras/Raf/MEK/ERK signalling pathway is crucial in cancer !!! • Genetic abnormalities found • in FGFRs that participate in cancer: • Chromosomal translocations • Gene amplification and overexpression • Activating mutations Changes in gene expression and cell response

14. I. Signals of single growth factor have different effects in two different cell types with respect to the different phenotype of these cells FGF DI DI FGF/FGFR = Ras-dependent signalling pathway DII DII HSPG DIIIb/IIIc DIIIb/IIIc II. P TK TK III. P TK TK P P IV. P Various MM isoforms of FGF-2 (I. - IV.) P • Cancer (transformed) stem cells • Resistance to drugs • Cell survival • Tumorigenicity • Differentiation • Self-renewal • Embryonic stem cells • Self-renewal • Differentaition • Cell survival

15. Harmatomas TSC1/TSC2 mTOR The possibility of genetic changes at multiple levels during signal transduction

16. …and this applies mainly for Ras kinase: Incidence of RAS mutations in human cancer

17. Mutations in the tyrosine kinase domain of FGFR2 and thus aberrant downstream signalling cause a variety of craniosynostosis syndromes Arg678Gly Ligand-binding domains Glu565Ala Glu565Gly Gly663Glu Cell membrane Asn549His Lys641Arg IgI IgII IgIII TK1 TK2 TM Computed tomography Cytoplazm Crouzon syndrome Pfeiffer syndrome

18. Examples from the laboratory and relationships to clinics: Fibroblast growth factors and their receptors regulate self-renewal and differentiation of stem cells Inhibitor Control Inhibition of phosphorylation of kinase domains of FGFRs by synthetic inhibitor SU5402 leads to rapid DIFFERENTIATION Differentiation The same inhibitor can be used in patients to inhibit aberrant FGFR downstream signalling caused e.g. by point mutations Undifferentiated growth Knock-down of endogenous FGF using siRNA approach leads to rapid differentiation of stem cells and their increased sensitivity to various stress conditions Similar strategy can be potentially applied to stimulate differentiation or inhibit growth of cancer stem cells

19. Example: FGFs and their receptors in early development Fibroblast growth factors function as morphogens and thus the inactivation of one of the high affinity receptors causes developmental abnormalities (1) One receptor missing All four FGFRs present (2) Since the concentration of morphogens (morhpogen gradient) regulates morphogenesis, any defect in signal transduction that mimics morphogen gradient causes abnormal development !!! • Skeletal displasias • & activating mutations • Mutation in FGFR1,2 or 3 & various craniosynostosis syndromes • Mutation in FGFR3 & achondroplasia Normal development Abnormal development

20. Fibroblast growth factors and their receptors regulate the behaviour of leukaemia cells Example – high concentrations of soluble FGF-2 in peripheral blood of patients with leukaemia can cause hyperproliferation of leukaemia cells and neoangiogenesis Sex/Age Stage Treatment FGF-2 (pg/ml) M/58 chronic no 14 M/59 chronic yes 30 M/50 chronic yes 63 M/42 chronic yes 11 F/49 chronic yes 96 M/78 chronic no 8 F/60 chronic yes 78 F/46 chronic no 2 F/35 chronic yes 64 M/55 acceleration yes 1 F/64 acceleration yes 13 M/39 acceleration yes 17 F/73 blast crisis yes 52 M/51 blast crisis yes 0 F/64 blast crisis yes 69 M/39 blast crisis yes 65 Healthy individuals mean = 9 pg/ml mean = 33 pg/ml

21. Fibroblast growth factors and their receptors regulate the behaviour of leukaemia cells Example –leukaemia patients accumulate enormously high amount of FGF-2 in their CD34+ stem/progenitor cells where FGF-2 associates with FGFR1 and resulting complex is translocated into the nucleus. This can make cancer cells more resistant to treatment Chronic myeloid leukaemia FGF-2 K562 CD34+ T B M/M CD34- Western blotting and separation of cell subsets FGFR1 = 10nm FGF-2 = 5nm

22. Fibroblast growth factors and their receptors regulate the behaviour of leukaemia cells Example – expression of FGFR3 corresponds to the progression of disease after transplantation of stem cells. Increased expression of FGFR3 in cancer cells can cause their increased stimulation and proliferation Time from transplantation (days) BCR-ABL FGFR-3 -90 + 104 - 105 +82 - <10 +124 ++103 - 104 +165 ++ 104 +221 +++ 103 +275 +++ 104 - 105 Healthy individuals <10 * * Gene copy numbers per known concentration of RNA

23. Fibroblast growth factors and their receptors regulate the behaviour of leukaemia cells Example – expression of FGFR3 corresponds to the development of disease and its treatment and thus can be used for diagnosis FGFR-3 Patient Stage/burden Therapy CD34+ CD34- * 1. chronic/I IFN+C 31 3 2. chronic/I IFN 4 1 3. chronic/II IFN 16 3 4. chronic/III IFN+HU 2200 18 5. chronic/III HU 1970 3 6. acceleration HU 786 123 7. Blast crisis HU 292 105 8. chronic/III IFN+HU 245 14 /+89 days chronic/II STI571 1 0 9. chronic/III IFN+HU 688 1 /+270 days chronic/II STI571 4 1 * Gene copy numbers per known concentration of RNA

24. Model of action of fibroblast growth factors and their receptors in normal and leukaemia cells normal haematopoiesis leukaemia haematopoiesis CD34+ stem cell & progenitor compartment FL FL SF SF FGF-2 in cell nuclei IL-3 IL-3 secreted FGF-2 G-CSF G-CSF FGFR3 FGF-2 in blood CD34- terminal differentiation compartment

25. Basic terminology in growth factor and signal transduction in normal and cancer cell field: • Epo- erythropoietin, growth factor that stimulates erythropoiesis • G-CSF- granulocyte-colony stimulating factor • ATP- ribonucleotide adenosintriphosphate that participates in energy transfer • GDP- ribonucleotide guanosindiphosphate that participates in energy transfer • GTP- ribonucleotide guanosintriphosphate that participates in energy transfer • kinase- molecule with enzymatic (catalytic) activity • oncogene- gene/gene products with proven oncogenic potential • protooncogene- gene/gene products eventually with oncogenic potential • IFN- cytokine interferon, in treatment of leukaemia disease frequently used • in combination with cytarabine (C) • HU- hydroxyurea • STI571- synthetic inhibitor of BCR-ABL tyrosine kinase • IL-3- haematopoieticcytokine interleukin 3 • SF- haematopoietic cytokine „steel factor“ • FL- haematopoietic cytokine, ligand of flt3/flk2 receptors

26. This presentation will be available at: https://is.muni.cz www.med.muni.cz/biologie/ Education materials Medical Biology - lecture course (spring 2009)