The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound directly to tumor necrosis factor receptors (TNFR), and disturbed the TNFα/TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFα-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFα signaling and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis.

Granulin epithelin precursor (GEP) has been implicated in development, tissue regeneration, tumorigenesis, and inflammation. Herein we report that GEP stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo, and GEP-knockdown mice display skeleton defects. Similar to bone morphogenic protein (BMP) 2, application of the recombinant GEP accelerates rabbit cartilage repair in vivo. GEP is a key downstream molecule of BMP2, and it is required for BMP2-mediated chondrocyte differentiation. We also show that GEP activates chondrocyte differentiation through Erk1/2 signaling and that JunB transcription factor is one of key downstream molecules of GEP in chondrocyte differentiation. Collectively, these findings reveal a novel critical role of GEP growth factor in chondrocyte differentiation and the molecular events both in vivo and in vitro.

Objective-ADAMTS-7 and ADAMTS-12, two members of the ADAMTS (adisintegrin and metalloprotease with thrombospondin motifs) family, associate with and degrade COMP by binding to the EGF domain of COMP. Granulin-epithelin precursor (GEP) is a secreted growth factor that mediates tissue regeneration, tumorigenesis, and inflammation, and may play an important role in the pathogenesis of arthritis through mechanisms yet unknown. GEP also binds to the EGF domain of COMP. This study is to determine 1) whether there exists a protein interaction network between GEP, ADAMTS-7/-12 and COMP; 2) whether GEP interferes with the interactions between ADAMTS-7/-12 metalloproteinases and COMP substrate, including the cleavage of COMP; 3) whether GEP affects TNFα-mediated induction of ADAMTS-7/-12 expression and COMP degradation; and 4) whether GEP levels are altered during the progression of arthritis.Methods-Yeast two-hybrid, in vitro GST pull down and co-immunoprecipitation assays were used to 1) examine the interactions between GEP, ADAMTS-7/-12 and COMP, and 2) map the binding sites required for the interactions between GEP and ADAMTS-7/-12; Immunoflouresence cell staining was performed to visualize the sub-cellular localization of GEP and ADAMTS-7/-12; an in vitro digestion assay was employed to determine whether GEP inhibits ADAMTS-7/-12-mediated digestion of COMP; The role of GEP in inhibiting TNFα-induced ADAMTS-7/-12 expression and COMP degradation in cartilage explants was also analyzed.Results-GEP binds directly to ADAMTS-7 and ADAMTS-12 in vitro and in chondrocytes, and the four C-terminal TSP motifs of ADAMTS-7/-12 and each granulin unit of GEP mediate their interactions. Additionally, GEP co-localizes with ADAMTS-7 and ADAMTS-12 on the cell surface of chondrocytes. More importantly, GEP inhibits COMP degradation by ADAMTS-7/-12 in a dose-dependent manner through the following two mechanisms: a) competitive inhibition through direct protein-protein interactions with ADAMTS-7/-12 and COMP; and b) inhibition of TNFα-induced ADAMTS-7/-12 expression. Furthermore, GEP levels are significantly elevated in patients with either osteroarthritis or rheumatoid arthritis.Conclusion-Our observations demonstrate a novel protein-protein interaction network between GEP growth factor, ADAMTS-7 and ADAMTS-12 metalloproteinases, and COMP extracellular matrix protein. Furthermore, GEP is a novel specific inhibitor of ADAMTS-7/-12-mediated ¶

Unlike skin, oral gingival do not scar in response to tissue injury. Fibroblasts, the cell type responsible for connective tissue repair and scarring, are exposed to mechanical tension during normal and pathological conditions including wound healing and fibrogenesis. Understanding how human gingival fibroblasts respond to mechanical tension is likely to yield valuable insights not only into gingival function but also into the molecular basis of scarless repair. CCN2/connective tissue growth factor is potently induced in fibroblasts during tissue repair and fibrogenesis. We subjected gingival fibroblasts to cyclical strain (up to 72 hours) using the Flexercell system and showed that CCN2 mRNA and protein was induced by strain. Strain caused the rapid activation of latent TGFβ, in a fashion that was reduced by blebbistatin and FAK/src inhibition, and the induction of endothelin (ET-1) mRNA and protein expression. Strain did not cause induction of α-smooth muscle actin or collagen type I mRNAs (proteins promoting scarring); but induced a cohort of pro-proliferative mRNAs and cell proliferation. Compared to dermal fibroblasts, gingival fibroblasts showed reduced ability to respond to TGFβ by inducing fibrogenic mRNAs; addition of ET-1 rescued this phenotype. Pharmacological inhibition of the TGFβ type I (ALK5) receptor, the endothelin A/B receptors and FAK/src significantly reduced the induction of CCN2 and pro-proliferative mRNAs and cell proliferation. Controlling TGFβ, ET-1 and FAK/src activity may be useful in controlling responses to mechanical strain in the gingiva and may be of value in controlling fibroproliferative conditions such as gingival hyperplasia; controlling ET-1 may be of benefit in controlling scarring in response to injury in the skin.

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