Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose: Fibroblast-like synoviocytes (FLS) in the synovial intimal lining are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). These cells assume a tumor-like phenotype in RA, aggressively invading the extracellular matrix and producing cartilage-degrading proteases and inflammatory cytokines. The behavior of synovial fibroblasts is controlled by multiple interconnected signal transduction pathways involving reversible protein phosphorylation. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. The objective of this study was to define all of the PTP genes (PTPome) expressed in FLS and determine if any play a role in the rheumatoid synoviocyte phenotype
Methods: Comparative screening was conducted of the PTPome expression in FLS from patients with RA or osteoarthritis (OA) by qPCR. Cell permeable anti-sense oligonucleotides were used to suppress PTPs, such as SHP-2, and achieved 90% knockdown. Transwell FLS invasion assays were performed using Matrigel-coated inserts. FCS or PDGF were used as chemoattractants, and invasion was quantified by propidium iodide (PI) staining of insert membranes. Transwell FLS migration assays were carried out using FCS as a chemoattractant, and migration was quantified by staining cells with Celltracker green. FLS apoptosis was quantified by flow cytometry staining with PI and Annexin V. MMP and cytokine gene induction after TNF stimulation were quantified by qPCR. Western blotting of cell lysates using phosphospecific antibodies was used to assess activation of signaling pathways.
Results: FLS display abundant expression of genes belonging to all of the known subfamilies of PTPs. Of these, only PTPRK, PTPN11, PTPN14 and DUSP3 expression were increased in RA (n=11) compared to OA (n=10) FLS (p<0.05). Subsequent studies focused on PTPN11, which encodes SHP-2, because it is a well-documented proto-oncogene. SHP-2 knockdown in RA FLS with antisense led to increased basal apoptosis (162% increase, p<0.05) and impaired invasion (71% decrease, p<0.05) and migration (44% decrease, p<0.05) in response to FCS or PDGF. SHP-2-deficient RA FLS displayed decreased activation of focal adhesion kinase and mitogen-activated protein kinases, such as JNK, in response to PDGF. Knockdown of SHP-2 also significantly suppressed TNF-mediated induction of MMPs and adhesion molecules, including MMP-1 (79% decrease, p<0.05), MMP-2 (64% decrease, p<0.05), VCAM-1 (93% decrease, p<0.05), and Cadherin-11 (77% decrease, p<0.05). Decreased gene expression correlated with a dramatically reduced induction of JNK phosphorylation in cell lysates.
Conclusion: These findings demonstrate a novel role for the proto-oncogene SHP-2 as a key mediator of FLS function. SHP-2 promotes the invasiveness and survival of RA FLS and, due to its higher expression in RA compared with OA FLS, could contribute to the unique aggressive phenotype of the rheumatoid cells. Therefore, SHP-2 could be a novel therapeutic target for RA.
Disclosure:
S. Stanford,
None;
M. Maestre,
None;
B. Bartok,
None;
D. L. Boyle,
None;
H. Arnett,
Amgen, Inc.,
1,
Amgen, Inc.,
3;
T. Mustelin,
None;
G. S. Firestein,
None;
N. Bottini,
Amgen, Inc.,
2.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ptpome-profile-of-rheumatoid-arthritis-fibroblast-like-synoviocytes-a-novel-role-for-the-tyrosine-phosphatase-shp-2-as-a-modulator-of-invasion-and-survival/