Growth Factors
and
Cytokines
• Most important biologically active
group of molecules to be identified
• Generally small to medium sized
proteins and glycoproteins
• Mediate potent biological effects on
all cell types
• Involved in all physiological
processes
Cytokines
• Interleukins
• Interferons
• Cytotoxins
• Colony Stimulating Factors
• Growth Factors
• Suppressor, Inhibitory Factors
• Stimulate or inhibit
– Cell proliferation
– Differentiation
– Migration
– Adhesion
– Gene expression
– Secretion and action of other growth
factors
• Different growth factors share the
same biological effects
• Most show more than one property and
are able to mediate vast array of
biological functions (pleiotropic)
• Currently 100+ have been discovered,
20 different families based on
structural homology
• Not stored as preformed molecules
• Require proteolytic activation
• May need to bind to ECM for activity
and stabilization
• Synthesis is initiated by new gene
transcription
• Act by binding to cell surface receptors
• Important autocrine and paracrine
regulators of cell growth and function
• Names indicative of original location of
discovery, not range of potential
effects
• Characterized by short biological half
lives (PDGF, 2 minutes in blood for
example)
Transforming Growth Factor b
• Part of superfamily that includes BMP, inhibin,
activin and Mullerian substance
• Three separate TGF-bs discovered (TGF-
b1,2,3)
• Originally named for ability to cause reversible
change in phenotype of rat fibroblasts
• Discovered independently as cartilage inducing
factor, glioblastoma immunosuppressive factor,
myoblast differentiation inhibition factor,
epithelial growth inhibitor
• Synthesized as the COOH terminal
112 amino acids of a 390 amino
acid precursor (12.5 kDa)
• Precursors dimerize (25 kDa)
• Intracellular proteolytic cleavage
thought to occur by furin peptidase
• Active TGF-b is usually a
homodimer, but naturally occurring
TGF-b1b2 and TGF-b2b3 have been
discovered
• Significant homology between
isoforms
– 98-100% homology between chicken
and human
– 70-80% homology between three
human isoforms
– Conservation of spacing between nine
cysteines
Latency and Activation
• Secreted from cells as inactive or
latent complex unable to bind receptor
• Mature dimer + two pro region peptides
(LAP) covalently linked
• LAPs can be disulfide linked to
glycoprotein 125-190 kDa – LTBP
• LTBP does not bind TGF-b directly but
may enhance release, activate latent
forms
Latency and Activation
• In vivo activation mechanism not known,
thought to be enzyme mediated
• In vitro
– Proteolysis by plasmin, cathepsin D
– Heat treatment
– Extremes in pH
• Clearance by binding to a2-macro
extremely rapid (<3 minutes)
• Transient and local availability
Cellular Sources and Production
• Most if not all mature cells produce at
least one TGF-b isoform
• Enhanced expression in the adult during
processes such as
– Tissue repair
– Bone remodeling
– Inflammation
• Stored at high levels in alpha granules of
blood
• Binds to fibronectin at sites of injury
Biological Activity
• Proliferative responses
• Cell differentiation
• Effects on differentiated functions
• Responses involving ECM
• In vitro effects can be either positive
or negative depending on cell type,
state of differentiation and culture
conditions
Biological Activity
• Three isoforms generally exhibit
the same overall effects, usually
with distinct potencies
• Reports exist of differential
responses of cells to TGF-b1 and
TGF-b2 due to altered receptor
binding properties or alterations in
receptor specific intracellular
signaling components
Cell Proliferation
• Inhibitory to most cells,
particularly epithelial cells,
endothelial cells, lymphoid cells,
hematopoietic cells
• Reports are contradictory
• Growth promotion effects have
been noted, may be indirect
through production of mitogens or
receptors
Cell – Environmental Interactions
• Migration, chemotaxis, tissue
formation, repair, remodeling, wound
healing depend on interactions between
cells and tissues
• TGF-b regulates cell adhesiveness on
many levels
– Increased synthesis of ECM
– Control of matrix degrading proteases
– Increased expression of cell adhesion
proteins
Cell – Environmental Interactions
• Downregulates EGF receptors in
fibroblasts
• Several receptors in hematopoietic
cells are downregulated
TGF-b – Receptor Interactions
• Most cells have three types of TGF-b
receptors at cell surface that regulate
effects on growth and differentiation
TGF-b
1
Type I
Receptor 2
Type II
Receptor
TGF-b – Receptor Interactions
• Both TbR-I and TbRII are needed
to generate activated receptor
complex
• Biological effects of TGF-b
mediated by type III receptors are
unknown
Cell Cycle Arrest
• Inhibitory effects marked by inability
of cells to enter S phase
Interactions with Proteins and
ECM
• Binds with high affinity to
– Type IV collagen
– Fibronectin
– Proteoglycans
– Serum proteins
• Binding to fn, collagen do not affect
activity
• Binding to some serum proteins results
in rapid neutralization
Epidermal Growth Factor
• Most characterized growth factor
• 53 amino acids, 6 kDa
• Stimulatory for wide variety of cell
types
• Initial changes include
– Increase in active transport of low MW
compounds
– Protein phosphorylation
– Membrane translocation
– Receptor internalization
EGF diagram
The EGF Receptor as a Model
Receptor Ligand Binding
• Often monitored using 125I
• Incubation of cells with ligand for
specified time
• Rapid removal of unbound ligand
• Measurement of radioactivity
• Non specific binding is measured by
adding high concentrations of
unlabeled growth factor to system
Specific binding diagram
Receptor + Ligand diagram
kf
R LC
krkf
R L C
kr
kr
KD
kf
RL
C
KD
• KD is equilibrium dissociation
constant
• Small KD, high KA (KD-1), equilibrium
association constant, means high
affinity of receptor for ligand
• High affinity KD = 10-15
• Low affinity KD = 10-6
• Function of temperature, pH
Receptor Mobility
• Coupling receptors with effector
molecules, other receptors,
cytoskeletal elements and additional
membrane associated components
requires diffusion within lipid
bilayer
Receptor Ligand Trafficking
Receptor Downregulation
• Can lead to receptor downregulation
• Essentially loss of cell surface
receptors
– Endocytotic (internalization step)
– Sorting
– Synthetic