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Conserved ligands, cell-surface receptors and signaling co-receptors from C. elegans to humans 3 subfamilies: TGF-b, activin/inhibin/nodal, BMP Ligands are both homodimers or heterodimers with a set of 7 cysteines: 3 intrasubunit disulfide bonds + 1 intersubunit bond

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tgf b superfamily

Conserved ligands, cell-surface receptors and signaling co-receptors from C. elegans to humans

3 subfamilies: TGF-b, activin/inhibin/nodal, BMP

Ligands are both homodimers or heterodimers with a set of 7 cysteines:

3 intrasubunit disulfide bonds + 1 intersubunit bond

Two types cell surface receptors: serine-threonine kinases= type I and type II Receptors

All type I receptors have a TTSGSGSG motif, called the GS domain

4 mammalian type II receptors: constitutively active

7 type I receptors= activin receptor like kinases 1-7 (ALK1-7)

A TGF ligand signals by binding to and bringing together type I and type II receptors to form a ternary holo-complex. The assembly dynamic differs between ligands

In the holo-complex, constitutively active type II kinase transphosphorylates the GS domain of type I, which then phosphorylates Smads

8 Smads, with 3 functional classes: receptor regulated Smads=R-Smad (1,5,8//2,3), co-mediator Smad= co-Smad (4), inhibitory Smad=I-Smad(6,7)

TGF-b Superfamily
tgf b superfamily2
TGF-b Superfamily

8 Smads, with 3 functional classes: receptor regulated Smads=R-Smad (1,5,8//2,3), co-mediator Smad= co-Smad (4), inhibitory Smad=I-Smad(6,7)

After phopshorylation, 2 or 3 activated R-Smad di-tri-merize and form heterometric complexes with smad4.--->nucleus

the I-Smads negatively regulate the signaling by competing with R-Smads for the receptors or co-Smad interaction and by targeting the receptors for degradation

Signaling through non-Smad pathways:

MAPK and ERK, p38, JNK, PI3K/Akt pathway, NF-kB pathway

tgf b signalling is regulated at all levels
TGF-b signalling is regulated at all levels

TGF-bs are secreted as inactive precursor proteins

BMPs are secreted in their active form, but activity is regulated by ligand antagonists:

3 families of BMP antagonists based on the size of their cysteine knot

TGF-b co-receptors:

-enhance ligand-binding to the receptors

-antagonize ligand function by shedding of extracellular domains

-regulate receptor localization and internalization

-regulate cell adhesion

-regulate signalling

Pseudo-Receptor

FK506-binding protein 12 (FKBP12), binds the GS domain of type I receptors and maintains them inactive.

Localization and trafficking of the signaling receptors: SARA or co-receptors.

TGF-b receptor trafficking and turnover

extracellular antagonists and membrane level regulation
Extracellular Antagonists and Membrane Level Regulation

Noggin:BMP-7 complex

Follistatin:activin complex

tgf b activation
TGF-b activation

The 3 TGF-b isoforms are synthesized and homodimeric pro-TGF-b = TGF covalently linked to latency associated protein (LAP)

Intracellularly cleaved by furin-type enzymes--> mature TGF-b , remains non-covalently associated with LAP= SLC

Few cells produce and secrete diffusible SLC. Majority secrete TGF as part of the LLC, formed within secretory vesicles: covalent disulfide bond between the SLC and a member of the LTBP family

LTBP-1,-3,-4 bind TGF-bs.

LTBPs belong to the superfamily of fibrillin-like ECM proteins

Bind a series of ECM proteins

TGF-b is synthesized in excess, and bioavailability depends on activation

latent tgf b activation

Activation of TGF-b varies according to cell type and physiological context

Heat, acid, reactive oxygen species, glycosidases, thrombospondin, proteolysis and integrin- mediated activation

Proteases:

Plasmin, thrombin, elastase, MT-MMP,MMP-2, MMP3, MMP-9, MMP13 etc.. in vitro. In vivo, however, simple proteolysis has not been shown. No supporting genetic evidence..

Predominantly non-proteolytic mechanism for TGF-b activation in vivo:

Integrin (av, b6, b8) and TGF-b KO mice have overlapping phenotypes.

The TGF-b1 RGD->RGE mutation phenocopies the TGF-b1 KO.

Integrins shown to bind and possibly activate latent TGF-b1

avb1: binding yes, activation not known.

avb3: binding yes, activation yes.

avb5: binding yes, activation yes.

avb6: binding yes, activation yes.

avb8: binding yes, activation yes.

a8b1: binding yes, activation no

xb1: binding yes, activation yes.

Latent TGF-b activation
latent tgf b activation10

Integrins bind to the RGD sequence at the N-term of LAP.

Mutation to RGE abolishes binding and activation in epithelial cells

RGD peptides reduce TGF-b activation in myofibroblasts

The TGF-b1 RGD->RGE mutation phenocopies the TGF-b1 KO.

avb6 has a higher affinity for the RGD in LAP than in FN due to a DLXXL motif.

Some LTBPs also have an RGD, but it has not been shown to be an integrin ligand.

Why do not all integrins that bind to the RGD in LAP activate TGF-b1??

Integrins shown to bind and possibly activate latent TGF-b1

avb1: binding yes, activation not known.

avb3: binding yes, activation yes.

avb5: binding yes, activation yes.

avb6: binding yes, activation yes.

avb8: binding yes, activation yes.

a8b1: binding yes, activation no

xb1: binding yes, activation yes.

Latent TGF-b activation
latent tgf b activation integrin dependent proteolysis
Latent TGF-b activation: integrin dependent proteolysis

Incubation of purified integrins with TGF-b is not sufficient to activate the latent complex.

Mechanism A: integrin docks latent TGF-b and a protease and brings them close together

avb8 said to mediate LAP-b1 activation in conjunction with MT-1 MMP: MMP inhibitor + co-localization

avb3 activation coincides with MMP-9 and MMP-2 production in cancer cells

Direct interaction of avb3 and TGF-b-RII upon TGF-b application has been shown by BRET

latent tgf b activation integrin mediated cell traction
Latent TGF-b activation: integrin-mediated cell traction

Mechanism B: traction forces due to integrin binding to latent TGF-b results in a conformational change that liberates TGF-b.

avb6, avb5, and possibly avb3 can activate Latent TGF-b independently of proteases

LAP-b1 has 2 conformations (far UV dichroism spectroscopy): only 1 binds to TGF-b1

There needs to be

An actin cytoskeleton

Specific integrins

Incorporation of LLC into the ECM

A mechanically resistant ECM

latent tgf b activation integrin mediated cell traction13
Latent TGF-b activation: integrin-mediated cell traction
  • A)Direct activation of latent TGF-b by integrins require a contractile cytoskeleton.
  • avb6: disruption of actin cytoskeleton with cytochalasin D or cytoplasmic truncation prevents latent TGF-b activation
  • Mefs with constitutively active RhoA activate latent TGF-b more. (less with dominant neg)
  • Highly contractile fibroblast but not low contractile fibroblasts release active TGF-b from the same LLC-rich ECM.
  • Inducing myofibroblast contraction with thrombin, angiotensin-II and endothelin-I increases TGF-b activation: integrin-dependant and protease-independant
  • Myofibroblast contraction blockers (ML-7, blebbistatin, cytochalasin, SMA-FP) inhibits TGF-b activation by contraction-inducers
  • External stretch of the myofibroblasts also activates latent TGF-b activation
  • C)LAP-b1 must be associated with LTBP in the LLC and be incorporated into the ECM for activation of TGF-b by integrins
  • Deletion of the ECM-binding hinge region of LTBP-1 (or substitution by LTBP-3 which lacks the hinge) abolishes avb6 integrin-mediated activation of latent TGF-b
  • Expression of an engineered LTBP with only the ECM-binding hinge region and the LAP binding domain is sufficient to promote avb6 integrin-mediated activation of latent TGF-b
  • different LTBP isoforms possibly regulate latent TGF-b activation
  • Does the matrix composition do as well???
latent tgf b activation integrin mediated cell traction14
Latent TGF-b activation: integrin-mediated cell traction

D)the efficiency of latent TGF-b activation by integrins depends on the mechanical properties of the ECM

Tissue culture dish=rigid substrate

Lung tissue of living mice: PAR1-activating peptides are not sufficient for TGF-b activation. Lungs must be mechanically challenged by hight-tidal-volume ventilation+ PAR1-activating peptides

Myofibroblasts activate latent TGF-b by integrin-mediated contraction when cultured on stiff silicone substrates, but not on silicone substrates that have the compliance of normal connective tissue.

latent tgf b activation15
Latent TGF-b activation

Activation of TGF-b varies according to cell type and physiological context

Heat, acid, reactive oxygen species, proteolysis and integrin mediated activation

Proteases:

Plasmin, thrombin, elastase, MMP-2, MMP-9 in vitro.

In vivo,

tgf b and angiogenesis
TGF-b and Angiogenesis

Loss of TGFb component->abnormal differentiation and maturation of primitive vascular plexus

TGFb1-> 50% E9-E10 lethality due to defects in yolk sac vasculature and hematopoietic system. Vessels with decreased wall integrity

TGFbRII, ALK5, ALK1, endoglin and Smad5 ->idem

However, TGFb1 and TGFbRII are critical for formation of the vascular plexus and remodeling into a complex network. ALK1, ALK5 and Eng only for angiogenesis

Endolethial-specific KO of ALK5 and TGFbRII phenocopy the complete KOs

tgf b and angiogenesis20
TGF-b and Angiogenesis

TGF-b1 has bifunctional effects on endothelial cells in vitro. It can both stimulate and inhibit proliferation