Dysregulaon of the!ADAM17/Notch! signaling!pathways!in ... · Iñaki&González Foruria,...

Iñaki González-‐Foruria, Pietro Santulli, Sandrine Chouzenoux, Francisco Carmona, Charles Chapron, Frédéric Ba@eux

Dysregula+on of the ADAM17/Notch signaling pathways in endometriosis correlates with disease severity and

fibrosis

Introduc+on

•  Benign gynaecological disease

•  Chronic inflammatory condi+on (Bulun, 2009)

•  Overproduc+on of prostaglandins, metalloproteases, cytokines and chemokines (Santulli, 2014)

•  Oxida+ve stress plays an essen+al role in the onset and progression (Ngo, 2009; Santulli, 2015)

Introduc+on

•  Oxida+ve stress induces the synthesis of inflammatory mediators

•  ADAM family metalloproteases, such as ADAM17 (Zhang, 2001)

ROS ADAM-‐17

Introduc+on

•  Oxida+ve stress induces the synthesis of inflammatory mediators

•  ADAM family metalloproteases, such as ADAM17 (Zhang, 2001)

•  ADAM family members are proteoly+cally ac+ve over target proteins, such as Notch (Milleri, 2013)

•  Notch are transmembrane receptors that regulate many cell processes (Hori, 2013)

ADAM-‐17

Notch complex

Mul+ple cell

func+ons

ROS

Introduc+on

•  Notch ac+vates trancrip+on of nuclear target genes related to fibrosis

•  Notch promotes fibroblasts prolifera+on and fibrosis (Kavian, 2014)

ADAM-‐17

Notch complex

Transcrip+onal ac+va+on

Mul+ple cell

func+ons

FIBROSIS

ROS

Nuclear transloca+on

Introduc+on

•  Notch ac+vates trancrip+onal regula+on of nuclear target genes related to fibrosis

•  Notch promotes fibroblasts prolifera+on and fibrosis (Kavian, 2014)

•  No data evaluates ADAM17/Notch signaling pathway in endometriosis

•  Hyperac+va+on of ADAM/Notch in other immunological diseases (Kavian, 2010)

Objec+ves

Methods

•  202 women, younger than 42 years-‐old, excluding cancer

•  Laparoscopy for gynaecological reasons

•  Endometriosis rAFS classifica+on

•  Endometriosis 3 phenotypes: –  Superficial (SUP) –  Endometrioma (OMA)

–  Deep infiltra+ng endometriosis (DIE)

Methods

•  Peritoneal fluid was obtained (n= 202)

•  Stromal endometrial cells collec+on and culture: –  Controls (n=8): endometrial biopsy (Cs)

–  DIE pa+ents (n=8): endometrial biopsy (Es) + DIE biopsy (Ps)

Methods

•  Advanced Oxida+on Protein Products –AOPP-‐ measurement in PF (Witko-‐Sarsat, 1998)

•  ADAM17 metalloprotease ac+vity in PF (ELISA) and cultured stromal endometrial cells (Cs, Es, Ps) (WB)

•  Notch ac+va+on (Notch intracellular domains -‐NICD-‐) in Cs, Es and Ps (WB)

•  Fibroblasts ac+va+on (α-‐SMA and type-‐I collagen) in Cs, Es and Ps with/without DAPT (WB)

Patient characteristics! Endometriosis (N=121) Controls (N=81)

P!Age (years) a 30.8 ± 5.1! 31.7± 5.36! 0.267t!Height (cm) a 167.5 ± 6.1! 164.1 ± 6.0! <0.001t!Weight (kg) a 59.5 ± 8.1! 61.5 ± 9.9! 0.162t!BMI (kg/m2) a 21.2 ± 2.5! 22.8 ± 3.4! 0.001t!Parity a 0.2 ± 0.5! 0.4 ± 0.7! 0.093t!Gravidity a 0.4 ± 0.7! 0.6 ± 1.0! 0.123t! rAFS Classification:!

Mean implants score rAFS a, d 11.3 ± 11.2! NA! !Mean adhesions score rAFS a, d 9.6 ± 16.3! NA!Mean total score rAFS a, d 21.2 ± 23.1! NA!rAFS stage (n, %): d

!

NA!

I

37( 30.6%)

II

16 (13.2%)

III

26 (21.5%)

IV 42 (34.7%)

Surgical classification: !Superficial endometriosis (n, %) 41 (33.9%)! NA! !Endometrioma (n,%) 32 (26.4%)! NA!

Endometrioma size (cm): a

!

!

Right

4.9 ± 2.8

NA

Left 4.8 ± 3.1

Endometrioma laterality (n, %): !

NA !

Bilateral

7/32 (21.9%)

Right

11/32 (34.4%)

Left 14/32 (43.7%)

DIE lesions (n, %) e 48 (39.7%)! NA! Mean number of DIE lesions a 2.3 ± 1.5! NA!

Total number of DIE lesions (n, %):

!

NA!1

13/48 (27.1%)

2

11/48 (22.9%)

≥3 24/48 (50.0%)

Anatomical distribution of DIE (n, %): e, b

!USL

35/48 (72.9%)

NA

Vagina

15/48 (31.2%)

Bladder

7/48 (14.6%)

Intestine

23/48 (47.9%)

Ureter 2/48 (4.2%)

Results

Results

•  AOPP levels and ADAM17 ac+vity are increased in endometriosis PF

Results

•  AOPP levels and ADAM17 ac+vity in PF present a posi+ve correla+on

Results

•  ADAM17 ac+vity is up-‐regulated in PF of DIE pa+ents

Results

•  ADAM17 ac+vity and Notch are up-‐regulated in Es and Ps with respect to Cs

Results

•  Increased Notch ac+vity in endometriosis pa+ents leads to a major ac+va+on of fibroblasts

Results

•  The use of DAPT to prevent NICD release, reduces fibrosis markers in Ps cells, but not in Cs cells

Conclusions

•  AOPP and ADAM17 increase and correlaYon in endometriosis

•  ADAM17 up-‐regulated in DIE

•  ADAM17 and Notch are hyperacYvated in stromal endometrial cells from DIE compared to control endometrial cells

Conclusions

•  Increased fibrosis in endometrial cells from DIE

•  Notch inhibi+on diminishes fibrosis only in endometrial cells from DIE pa+ents, but not control cells

•  ADAM17/Notch signaling pathway is up-‐regulated leading to major fibrosis in endometriosis

[email protected]

*This work was supported by grants from University Paris Descartes, INSERM and Fundación Alfonso Maran Escudero.

Thank you for your a@enYon

ADAM-‐17

Notch complex


Transcrip+onal ac+va+on

FIBROSIS

ROS

Metalloproteinase ADAM-‐17 cleavage

NICD γ-‐Secretase complex

ROS ADAM-‐17

Notch complex

NICD γ-‐Secretase complex


NICD Transcrip+onal ac+va+on

FIBROSIS

Endometrial cells

NKG2D ligands cleavage

Soluble NKG2D ligands

Metalloproteinase ADAM-‐17 cleavage

NK cell inhibi+on

Dysregulaon of the!ADAM17/Notch! signaling!pathways!in ... · Iñaki&González Foruria,...

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