W11L2 Flashcards
Transforming Growth Factor (TGF) β basics
Transforming Growth Factor (TGF) β is a cytokine, discovered due to its ability to induce proliferation and growth of cells in culture
Works in a paracrine or autocrine fashion
The TGF-β superfamily consists of >30 structurally related polypeptide growth factors including:
- TGF-βs (1–3)
- activins (A, B)
- inhibins (A, B)
- bone morphogenetic proteins (BMPs 1–20)
- growth differentiation factors including myostatin, nodal, leftys (1,2)
- Müllerian-inhibiting substance (MIS)
Regulates embryonic development and cellular homeostasis, including apoptosis, proliferation, differentiation, and extracellular matrix remodeling in a cell and context specific manner
Is dysregulated in numerous cancers having both positive and negative effects of tumour progression
TGFβ signaling - steps in signalling
TGF-β binds TGF-β receptor II, which is a serine/threonine kinase receptors
TGF-β receptor II forms a heteromeric complex with TGF-β receptor I; active TGF- β receptor II phosphorylates and activates TGF-β receptor I. Also results in dimerization between receptor I and II
SARA is at TGF-β receptor I. SARA (SMAD anchor for receptor activation) recruits receptor regulated (R)- Smads to the complex; R-Smads are phosphorylated by TGF-β receptor I
Two activated R-Smads form a complex with the common Smad called Smad4
Smad4/R-Smad translocates to the nucleus and interacts with transcription factors, co-activators or co-repressors to modulate gene expression
Canonical TGFβ signaling involves SMAD translocation while non-canonical signaling can trigger other mediators such as MAPKs – p38, JNK and ERK
- non-canonical signalling does not need Smad4
- Smad4 is mutated in some cancers, which does NOT turn off TGFβ signaling, but rather switches it to non-canonical signalling, which has detrimental effects on the cell
Different sub-types of SMADs
Different sub-types of SMADs
- Receptor-regulated SMADs (R-SMADs) –
SMAD 1, 2, 3, 5, 8/9 - Common factor SMADs - SMAD4
- Inhibitory SMADS (I-SMADs) – SMADs 6 and 7
- binds to R-SMADs to keep them inactive
TGFβ signaling - pathway components
- Something on the surface to recognize the environment change
- TGFβ receptors on the surface - Existing networks of proteins that can rapidly switch from an active to inactive state
- TGFβ receptor complex and Smads are already present, activation triggers a series of phosphorylation events to activate the pathway - Mechanism for entering the nucleus quickly and efficiently
- Interaction between Smad 4 and R-Smads dimers drives nuclear localization - Genes that are poised to be activated (or repressed)
- Smads bind to existing transcriptional regulators to affect gene expression
Bone morphogenetic protein vs. TGFβ signaling
- TGF-β is synthesized and secreted as an inactive precursor protein; BMPs are secreted in their active form
- Activation of TGF-β1 involves proteolytic cleavage; BMP is regulated by antagonists which bind BMPs and prevent interactions with their respective type I and type II receptors (BMPRI and BMPRII)
- TGF-β signaling involves SMADs 2/3; BMPs involve SMADs 1/5/8
- They BOTH have Smad4 in the pathway thought
Involved in different developmental processes
- TGF-β signaling: cytostatic response, environmental modifying responses, phenotypic plasticity responses
- BMPs: bone and cartilage development, postnatal bone formation, embryonic development
TGF signaling regulation
Level of the ligand
- Natural inhibitors such as noggin, follistatin, chordin, CAN family
- Absence of proteases activating TGFβ
Level of the receptor
- FK506-binding protein 12 (FKBP12)
maintains type I receptor in an inactive
conformation
- receptor can ubiquitinated and
degraded
Level of Smads
- Smad2/3 binding to SARA retains them in the cytoplasm
- Presence of inhibitory (I) Smads 6/7
Level of Transcriptional complexes
- Sno and Ski bind repress SMAD complex gene activation
TGFβ signaling in Development
TGFβ signaling factors (ligands, inhibitors and receptors) are expressed throughout development in all tissues and are required for most developmental processes
- cell migration
- gastrulation
- cell growth and metabolism
- DV axis specification
- bone formation
- dendritogenesis
- axonal transport
- proliferation
– note: TGFβ signalling can also repress proliferation. Original experiments that showed TGFβ signalling induces proliferation were performed in cancer cells. In normal cells though, TGFβ signalling has different effect.
ALK – BMP receptors
TβR – TGFβ receptors
Single KOs show importance of signaling dose: TGFB1−/− mice and TGFB2−/− mice
TGFB1−/− mice
- embryonic lethal
- excessive systemic inflammatory response
- massive infiltration of macrophages and
lymphocytes into the heart and lungs
TGFB2−/− mice
- embryonic lethal
- ventricular septum defects (VSD),
myocardial thinning, and a double outlet right ventricle (DORV)
- Have multiple defects in most organs
examines
– enlarged inner neuroblastic layer, cornea thickening, Ventricle septal defect, Bifurcated sternum, more cartilage, more proliferation, lack of differentiation
- impacts development too. on day 10, the embryos look the same as wildtype. But on day 12, you can see that the KO is smaller, less blood development, and heart defect (endocardial cushion does not develop)
The fact that TGFB1/2 activate different pathways (Even though use similar proteins) shows that it is not redundant
Single KOs show importance of signaling dose: noggin(NOG)−/−
Growth plates are enlarged and joint
initiation is disrupted
The zone of polarizing activity and apical ectodermal ridge are developmental and allow patterning of the limb
- zone of polarizing activity tells what is on top or bottom. i.e. mutation may make all fingers similar since it does not know which one should be the thumb
- apical ectodermal ridge prevents early differentiation via signals. Your forearms and arms form before your hands
Mice with no Noggin show altered skeletal development in limbs, ribs and head
Are mutations in TGFβ signaling found in humans?
- Most complete loss of function mutations are lethal early in development
Marfan syndrome (MS)
autosomal dominant multisystem connective tissue disorder
cardiac abnormalities, skeletal manifestations, and vision problems
Tall, long limbs, short torso
Inherited disorder
linked to mutations in the fibrillin-1 gene
- Fibrillin normally stabilize latent TGF-β-binding proteins and maintain TGF-β in its inactive state
- mutant fibrillin proteins in MS patients fail to do so, resulting in elevated levels of active TGF-β
- mutations were identified in TGFBR1 and TGFBR2 in patients with MS
Loeys–Dietz syndrome
autosomal dominant disorder
- aortic aneurysm syndrome
- cleft palate, and widespread vascular dilation
- high risk for aortic dissection or rupture
- Skeletal abnormalities
- bilateral clubfoot
- fusiform aneurysm of the left subclavian artery (expanding of artery)
- diffuse tortuosity (twisted, not straight) of vertebral arteries
somatic mutations in TGFBR2 and TGFBR1
- increased TGF−β signaling in their aortic walls, suggesting that these mutations are either gain of function
SMAD4
Critical to both TGFβ and BMP signaling pathways
Germline mutations for SMAD4 linked to:
1. Juvenile polyposis
- results in increased polyps in the intestinal tract and this predisposes people to colon cancer
- SMAD protein complex is not activated and not transported to the nucleus
- Hereditary hemorrhagic telangiectasia syndrome
- Mutations exist in TGFβ receptors
- leads to vascular dysplasia and vascular bleeding - Myhre syndrome
- abnormally stable SMAD4 protein that remains active
- leads to increased fibrosis and multiorgan issues and intellectual disability
TGFβ signaling in Cancer
Mutating any of the proteins of the TGF-β Signaling Pathway in a somatic way, it will predispose the tissue to undergo transformation and become oncogenic
Appears that inhibition of TGFβ promotes cancer
Over-expression of DN-TβRII in the pancreas:
- Increased proliferation, but impaired maintenance of the differentiated state
- Inhibition of TGFβ after determination results in acinar cells reverting to a more progenitor-like state
Appears that loss of some components of TGFβ signaling may unlock other functions:
- In breast, colon, lung, head and neck, pancreas cancer, there is decreased TGFβ signaling
- BUT, in breast, colon, lung, and prostate cancer, there is Gain of TGFβ function
