TGF-b signalling

What components are required in a signalling pathway?

Ligand

Receptor

2nd messenger

Effectors

Inhibitors

Canonical TGF-b family signalling

Ligands act via cell surface serine/ threonine kinase receptors

Ligand binds type II receptor first, which induces its constitutive activity and phosphorylate type I receptor

Type I receptor gets phosphorylated in their GS domain

Activate kinase of type I receptor and serves as a binding site for R-SMADs

R-SMADs get phosphorylated and form complex with SMAD 4. Creation of a trimers which goes into the nucleus and induce gene expression

What are the SMAD protein structure?

R SMAD

Co-SMAD - SMAD 4

I-SMAD

R SMAD: MH1 - Linker - MH2 - SSXS

Co-SMAD - SMAD 4: MH1 - Linker - MH2

I-SMAD: Linker - MH2

Linker - site of phosphorylation by MAPK and interaction with ubiquitin ligase

MH1 domain = DNA binding domain

SMAD 4, 3, 1, 5 have DNA binding capabilities, SMAD2 DOES NOT

Binds GC rich DNA motif

How TGF-b family is regulated by pro-domains

All secreted ligands synthesised as pre-pro-proteins

pre and pro domains are often cleaved intracellularly, but pro domain often remains bound to mature ligand, which help regulate the mature ligand availability and keep it inactive until needed eg. LAP (latent associated peptide) for TGF-b

To be activated and binding to the receptor

They can be bind to the integrin a5b1,3,4,6,8 to remove the TGFb from the LAP by exhibiting cytoskeletal tension, releasing active TGF-b for binding

TGF-b family regulation by I-SMADs

I-SMADs - SMAD 6 and SMAD 7

Lack the MH1 domain found in R-SMADs and SMAD 4, lack the SS(V/M)S motif in R-SMADs

Inhibit the pathway by

Binding to type 1 receptor and block R-SMAD binding, targeting the receptor for degradation

Binding to R-SMADs and preventing complex formation with SMAD4

Smad 6 prefers to inhbit BMP arm even though it can have effect on TGF-b

Smad 7 can inhibit both arms effectively

What are the two main arms of the TGF-b family?

1. TGF-b/ Activn/ Nodal darm → signalling mainly via Smad 2/3

2. BMP / GDF arm → signalling mainly via Smad 1/5/8.

What does TGF-b family signalling do?

Wound healing - matrix deposition, cell proliferation, angiogenesis

Excessive TGF-b can induce fibrosis

Induce EMT and associated with stem cell properties

Induces bone formation

Suppressing excessive muscle development

Role of TGF-b in cancer: in the canonical TGF-b/ Activin/ Nodal arm activates Smad 2/3 complex (with Smad 4) which leads to the transcriptional upregulation of CDK inhibitors such as

p51

p21

Triggers G1 arrest

In early tumour formation, TGF-b and Activin are… howver in the later stage…

Tumor-suppressive

In a multistage mouse skin carcinogenesis model…

TGF-b1 inhibited initial papilloma formation (tumour suppressive) but TGF-b1 promoted the progression of papilloma to carcinoma

The role of pro-oncogenic TGF-b in late stage tumorigenesis

• TGF-b may activate non SMAD pathway in cells resistant to growth suppression

• TGF-b produced by tumours can act on stromal fibroblast which promotes a cancer associated fibroblast phenotype

Promote a wound-healing phenotype associated with matrix deposition, angiogenesis, recruitment of immunosuppressive immune cells

High tgf-b = resistant to immunotherapy

Promotes EMT/ associated with cancer cell migration

BMPR1A mutation - loss of function

In juvenile polyposis syndrome - which drives benign tissue growth in the colon that can becomes malignant

GREM-1 large upstream duplication in hereditary mixed polyposis syndrome (HMPS)

GREM-1 is an antagonist of BMP

BMP is a growth break in the intestinal epithelium

GREM-1 usually expressed at the base of crypts

BMP signalling is low at the base of the crypt and increase in the villus

GREM-1 enhancer duplication increases GREM-1 expression in HMPS patients and cause tumour in mice

Mutation in SMAD4?

Very common in many cancer types

Smad4 lost would inactivate both arms of TGF-b signalling

R-SMADs (receptor regulated)

SMAD 1,2,3,8/9

Directly phosphorylated by type I receptors (ALKs) after ligand binding — SMAD1/5/8 for BMP, SMAD2/3 for TGF‑β/Activin/Nodal

Co-SMAD

Smad 4

forms trimer with R-SMADs

nuclear translocation and DNA binding

I-SMAD

SMAD6, SMAD 7

Block receptor–R‑SMAD interaction or promote receptor degradation

Has NO DNA binding domain (NO MH1)!!