Abstract: TH-PO923
Cleavage of the Matricellular Regulators of Fibrosis, CCN2, and 3 at the Third Intermodular Junction
Session Information
- Molecular Mechanisms of CKD - I
October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 1903 CKD (Non-Dialysis): Mechanisms
Authors
- Dear, Abigail, South West Thames Institute for Renal Research, Surrey, United Kingdom
- Phanish, Mysore K., Epsom and St helier University Hospitals NHS Trust, Carshalton, London, United Kingdom
- Tam, Frederick W.K., Imperial College Kidney and Transplant Institute, London, United Kingdom
- Dockrell, Mark E., South West Thames Institute for Renal Research, Surrey, United Kingdom
Background
CCN proteins are modular matricellular factors with roles in cancer and fibrosis. Members of the family consists of 4 modules encoded by 4 exons. The strucuture contributes to their role regulating interactions between cells and matrix.
CCN2/CTGF is a fibrogenic factor with actions in the lung, liver, skin and kidney. CCN3 is reported to counterbalance the actions of CCN2. We have previsouly described the expression and anti-fibrotic effects of CCN3 on proximal tubule cells when we also identified a truncated form consisiting of just the first 3 modules.
Here we investigate the enzymatic truncation of both CCN2 & 3 and the resultant effects.
Methods
Human primary proximal tubules were cultured on collagen IV. Protein expression was determined by western blotting and immunofluorescent microscopy.
Bioinformatic analysis to identify potential cleavage sites was performed using expasy peptide cutter..
Mass spectrometry was carried out using 2D nano ES-MS/MS analysis after trypsin digestion.
Results
Enzymatic cleavage sites were identified between modules 3 & 4 of CCN2; two for glutamyl peptidase, and one each for HIV-1 retropepsin and MMP9. Accumualtion of secreted full-length CCN2 following TGFβ1 treatment dropped by 72 h following MMP9 induction. We hypotheised MMP9 was cleaving the protein. Co-incubation with the MMP9 specific inhibitor prevented the fall in full length molecule.
Full length hrCCN2 potentiated TGFβ-induced fibronectin expression while the 4th module of CCN2 reduced TGF β-induced fibronectin in primary PTEC in culture.
Enzymatic cleavage sites were identified between modules 3 & 4 of CCN3 including two for cathepsin K and one for MMP9.
Inhibition of MMP9 did not alter the amount of CCN3. Mass Spec analysis of the 39KDa CCN3 isoform also suggrested cathepsin K.Cathepsin K is known to be induced by members of the TGFβ family.
Conclusion
The enxymatic cleavage of CCN2 & 3 acts a postranslational modiification altering the nature of the proteins..Although the potentially opposing factors share a putative MMP9 cleavage site the actually cleavage is performed by different enzymes. The induction of cleavage appears to be part of an endogenous regulatory process by TGFβ.
Targeting the enzymatic cleavage of CCN proteins may be an alternative therapeutic approach in fibrosis and cancer.
Funding
- Private Foundation Support