The Tissue Engineer s Toolkit

The Tissue Engineer s Toolkit Stimuli Detection and Response Ken Webb, Ph. D. Assistant Professor Dept. of Bioengineering Clemson University

Environmental Stimulus-Cellular Response

Environmental Stimuli Soluble signaling molecules Hormones Growth factors Adhesion molecules Cell-cell Cell-matrix Material Structure / Mechanical stimulation

Six Steps in Cell Signaling (Soluble Factors) Signaling cell 1. Synthesis 2. Release 3. Transport Target cell 4. Binding 5. Signaling 6. Desensitization Enzyme Activity Gene Expression

Specificity of Signaling

Endocrine Signaling Hormones

Paracrine Signaling Neurotransmitters Growth factors

Autocrine Signaling Growth factors

Receptors Intracellular Ion-channel Surface G protein-linked Receptor with guanylyl cyclases Receptor with tyrosine kinases Enzyme-linked Receptor with serine/threonine kinases Receptor with tyrosine phosphatases Receptor w/o enzyme activity

Surface versus Intracellular Receptors

Intracellular Receptors Lipophilic-diffuse across plasma membrane and bind cytosolic receptors Cortisol Progesterone Testosterone Thyroxine Retinoic acid

Molecules with Intracellular Receptors Low molecular weight, hydrophobic

Gene Regulation by Intracellular Receptors

Early / Delayed Signal Responses Immediate / early genes Transcription factors

Gated Ion Channels

(Surface)Trans-membrane Receptors Extracellularligand binding domain Plasma membrane outside inside Ligand Transmembrane domain Intracellular domain Intrinsic enzymatic activity / Binding sites for adapters / enzymes

Receptor Characteristics Specificity in general, receptors recognize a single ligand and exhibit minimal non-specific binding High affinity receptors can effectively bind ligands present at very low concentrations (nm pm) Saturability Receptor number is limited and therefore only a finite amount of ligand can be bound. Reversibility ligand-receptor binding is non-covalent and reversible as ligand concentration decreases Coupling receptor intracellular domains either contain intrinsic enzymatic activity or binding sites for enzymes such that extracellular binding results triggers intracellular biochemical reactions

Signal Transduction Binding Change in conformation Change in enzymatic activity Amplification Integration Response Down-regulation Key Concept: Multi-step pathway creates multiple opportunities for regulation, integration, inhibition

Conserved Mechanisms in Signal Transduction Second messengers GTPase switch proteins Protein kinases / phosphatases Adapter / scaffold proteins

Second Messengers camp-cyclic AMP cgmp-cyclic GMP DAG-1,2-diacylglycerol IP 3 -inositol 1,4,5- triphosphate Ca ++ Primarily activated through G-protein receptors in response to hormones However, the activity of these signaling molecules can influence signal transduction by other mechanisms

GTPase Switch Proteins GTP-Guanosine triphosphate GDP-Guanosine diphosphate

Regulation of GTPase Proteins GAP-GTPase activating proteinsincrease rate of GTP hydrolysis and therefore inactivate Ras GEF-guanine nucleotide exchange factors-promote GDP release and binding of GTP, leading to activation of Ras GDI=guanine nucleotide dissociation inhibitor decreases GTPase activation by blocking interaction with GEF

Protein Kinases Contain a highly conserved kinase (enzyme) domain Kinase-transfers a phosphate group from nucleotide triphosphate (usually ATP) to specific amino acid residue containing free hydroxyl group Exhibit high substrate specificity Two types: Tyrosine Kinase Serine/Threonine Kinase

Tyrosine Kinases Tyrosine kinases include many growth factor receptors :Platelet-derived growth factor receptor(pdgfr) Epidermal growth factor receptor(egfr) Insulin receptor Insulin-like growth factor 1 receptor(igf1r) Stem cell factor (SCF) receptor

Serine / Threonine Kinases

Protein Kinases / Phosphatases

Phosphorylation Most common mechanism for regulation of protein function Phosphorylation alters the structural conformation of a protein, causing it to become activated, deactivated, or modifying its function Reversible reaction suitable for regulatory control Abnormalities common to many diseases Therapeutic target

Regulatory Effects of Phosphorylation Holmberg et al. (2002) Trends Biochem Sci 27:619-27

Multiple Phosphorylation Sites Allow fine-tuning of kinase / transcription factor activation and bioactivity Holmberg et al. (2002) Trends Biochem Sci 27:619-27

Kinase Expression is Tissue Specific Expression of src-family kinases Src Ubiquitous Fyn Ubiquitous Yes Ubiquitous Lyn Hck Fgr Blk Lck Brain, B-cells, myeloid cells Myeloid cells Myeloid cells, B-cells B-cells T-cells, NK cells, brain

Adapter Proteins

Tyrosine Kinase Domains SH=src homology-regions that mediate protein-protein binding Facilitate kinase binding and accelerate signaling reactions

Signaling Scaffolds

Signaling Scaffolds

Signal Amplification

Second Messengers versus Enzymes Second messengers are very low molecular weight, diffuse rapidly and can act at multiple sites within the cell Kinases act more locally, commonly physically immobilized to plasma membrane, signaling scaffolds, or the cytoskeleton

GTPases versus Kinases GTPase activity is regulated by GTP / GDP binding, hydrolysis of GTP leads to inactivation Kinases catalyze ATP hydrolysis and the addition of released phosphate groups to other proteins / enzymes

G Protein-coupled Receptors

Tyrosine Kinase-linked Receptors

G-Protein Coupled Receptors (GPCR)

GPCR Ligands Hormones: Glucagon, Leuteinizing hormone, and Thyroid stimulating hormone Neurotransmitters: Epinephrine and norepinephrine Local mediators: Bradykinin, and Vasopressin

G Protein Signal Transduction Receptor binding G Protein activation Enzyme activation Second Messenger generation Target enzymes Transcription factors

Receptor Activation of Adenylyl Cyclase Activated receptor functions as a guanine nucleotide exchange factor (GEF) activating associated G protein

Adenylyl Cylase

Generation of camp

Activation of Protein Kinase A by camp *PKA : camp-dependent protein kinases, Serine/Threonine kinase *Activated PKA: phospholylates specific substrate protein molecules => Cellular response: rapid response: glycogen metabolism (~ seconds) slow response : gene transcription (~hours)

Regulation of blood glucose level

PKA Regulates Glycogen Metabolism PKA increases glucose level in blood by i) breaking down glycogen to glucose by activating phosphorlyase ii) Inhibiting glycogen synthesis by inactivating glycogen synthetase

PKA Regulates Gene Expression CRE:Cyclic AMP Response Element CREB: CRE-binding protein CBP:CREB-binding protein transcriptional coactivator

Amplification of GPCR Signaling

Enzymatic Regulation of Inositol Lipids

Activation of Inositol Lipid Signaling PI = phosphatidyl inositol

Ca-calmodulin dependent kinases Myosin light chain kinase

Receptor Tyrosine Kinases

RTK Activation Receptor Dimerization

RTK Activation PDGF: a dimer => directly cross-link two receptors together FGF: monomer => binds in clusters in proteoglycans => cross-link two receptors and phosphorylate each other Ephrins: a membrane bound protein in a signaling cell => Ephrins cluster in the plasma membrane, so cross-link their receptor

RTK to Ras Signaling Ras contains a covalently attached lipid group that serves to anchor the protein to the cytoplasmic face of the plasma membrane Two cytosolic proteins: GRB2 :functions as an adaptor protein Sos: functions as guanine nucleotide excahnge protein (GEF) Convert inactive GDP- bound Ras to active GTP-bound Ras

RTK Activation of Ras SOS

Activation of MAPK cascade by RTK pathway Raf: MAP-kinase-kinase -kinase MEK: MAP-kinasekinase MAPK-MAP kinase Mitogen activated protein kinase aka ERK-extracellularregulated kinase

MAPK Cascade EGFR FGFR PDGFR Trk A/B MAPK=mitogen-activated protein kinase

MAPK Stimulates Cell Division Phases of Eukaryotic Cell Cycle

Checkpoints in the Cell Cycle

Cyclin-cdk Complexes Regulate Cell Cycle Cdk 4 and cdk6 are both MAPK/ERK targets cdk-cyclin-dependent kinase

Activation of Cyclin / cdk Complex

Mitogen Stimulated Cell Cycle Progression

Cyclin-cdk Activity is Regulated by Inhibitors

Many RTKs Promote Cell Survival RTK Signaling Blocks Apoptosis Apoptosis-programmed cell death Cytoskeletal collapse Proteolysis of cytoplasmic proteins Disassembly of nuclear envelope DNA fragmentation

Apoptosis by caspases Initiator Capases Effector Caspases * Caspases: A family of proteases responsible for apoptosis Synthesized as inactive precusors, procaspases Procaspases are activated by other active caspases and cellular stresses

Two Mechanisms of Apoptosis Extrinsic Pathway-Procaspases activtion triggered from outside the cells Intrinsic Pathway- Procaspases activation triggered from within the cells

Apoptosis-Extrinsic Pathway

Apoptosis-Intrinsic Pathway

Phosphatidyl inositol-3-kinase (PI-3-K) Catalyzes phosphate addition from ATP to PIP 2, generating PIP 3 Contains 2 SH2 domains for binding and activation through RTKs Critical role in promotion of cell survival through activation of protein kinase B (PKB) aka Akt

Phosphatidyl Inositol-3-Kinase

PI -3-Kinase Signaling Prevents Apoptosis :Bcl2 PDK1: Phosphatidyl inositol -dependent protein kinase PKB: Protein kinase B (Akt) BAD(apoptosis promoter ): hold death- inhibitory protein such as bcl2

BAD and Bcl2 BAD-pro-apoptotic, if free and activated, binds to mitochondrial membrane and increases permeability-cytochrome c release, intrinsic pathway of apoptosis Bcl2-anti-apoptotic, reduces mitochondrial membrane permeability

Signaling Down-regulation Pathway regulation converging inhibitory pathways sequestration of intermediaries Receptor modulation internalization degradation inactivation Inactivation-hydrolysis of cyclic nucleotides / phosphatases Intrinsic short half-life of second messengers

Inhibition of GPCR Signaling

camp Regulation Activated by Ca-calmodulin

Inhibition of GPCR Signaling Activity

Down-regulation of Inositol Lipid Signaling 1. IP 3 rapidly dephosphorylated by phosphatases. 2. DAG rapidly hydrolyzed. 3. Ca 2+ rapidly pumped out. 4. Ser/Thr phosphatases dephosphorylate PKC and CaM kinase targets.

Negative Control of Signaling

Negative Control of Signaling

Mechanisms Regulating Signal Specificity Cell-type specific receptor expression levels Variation in cell-type specific kinase / phosphatase expression Variation in adapter proteins Cross-talk with other signaling mechanisms-adhesion / force