To explore further the anti-inflammatory mechanisms of PGRN and Atsttrin, we investigated
the role of PGRN and Atsttrin in the TNFa-
induced activation of IKK (IkB kinase)–IkB–
NF-kB (nuclear factor kB) signaling. rhPGRN
and Atsttrin blocked TNFa-induced phosphorylation of IKK and IkBa and the degradation of
IkBa in BMDMs (Fig. 6A). We also found increased IkBa phosphorylation in the tarsal joint
articular cartilage of mice with CIA, which was
abolished by treatment with rhPGRN or Atsttrin
(Fig. 6B). Treatment of BMDMs with rhPGRN
or Atsttrin impaired TNFa-induced NF-kB nuclear translocation, NF-kB binding to the IkBa
promoter, and activation of gene expression by
NF-kB (Fig. 6, C to F) (33). rhPGRN and Atsttrin
also inhibited the TNFa-induced phosphorylation of p38, JNK (c-Jun N-terminal kinase), and
ERK1/2 (extracellular signal–regulated kinase 1/2),
mitogen activated protein kinase (MAPK) family
members known to play an important role in
TNFa-mediated inflammation (Fig. 6G). Whereas
Atsttrin completely blocked the TNFa-induced
phosphorylation of ERK1/2, the presence of
rhPGRN resulted in only a partial inhibition of
this pathway. This may not be surprising, however, because PGRN itself has been previously
shown to activate ERK1/2 signaling (3). Taken
together, these results demonstrate that PGRN
and Atsttrin inhibit TNFa-induced intracellular
Collectively, our findings support the notion
that PGRN is a key regulator of inflammation
that may exert its anti-inflammatory effects, at
least in part, by blocking TNF binding to its
receptors. Whether this mechanism accounts for
all of the anti-inflammatory effects we observed
remains to be further elucidated.
During inflammation, neutrophils and macrophages release proteases that digest PGRN into
individual 6-kD granulin units, which are actually proinflammatory and can neutralize the
anti-inflammatory effects of intact PGRN (7, 8).
PGRN’s anti-inflammatory actions are protected
by its binding proteins, which include the secretory
leukocyte protease inhibitor (8) and apolipo-protein A1 (34 ), both of which bind to PGRN and
protect it against proteolytic degradation. Consistent with these observations, we found that the
PGRN-derived protein, Atsttrin, exhibits highly
potent anti-inflammatory activity that, in vivo, sur-passes that of PGRN itself. This occurred despite
the observation that PGRN binds to TNFR with a
higher affinity than Atsttrin. This may be because
Atsttrin contains only partial granulin units and
would not be expected to release any intact proinflammatory granulin units upon exposure to
PGRN-converting enzymes such as elastase (8),
proteinase-3 (7), and ADAMTS-7 (35). Moreover, Atsttrin exhibited a substantially longer half-life (~120 hours) than PGRN (~40 hours). The
identification of PGRN and the PGRN-derived
protein, Atsttrin, as antagonists of TNFR may lead
to innovative therapeutics for various pathologies
and conditions, such as rheumatoid arthritis.
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36. We thank P. Lengyel, J. Vilcek, and T. C. Caskey for
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G. Wisniewski for assisting with the CIA model, M. E. Dorf
and A. Mukundan for reagents, and A. Martin and
J. Quinn for assistance in Surface Plasmon Resonance
assay. This work was funded by NIH grants AR050620
and AR053210 (to C.-J.L.), GM061710 (to A.-H.D),
AI43542 (to M.L.D), and AR040072 (to J.C.); by a grant
from Arthritis National Research Foundation (to C.-J.L.);
by a National Nature Science Foundation of China
Grant 30870121 (to X.P. Y); and by a National Outstanding
Young Scientist Award of NSFC 30725015 (to Z.N. Y).
Patents have been filed by NYU that claim peptides
targeting TNF family receptors and antagonizing TNF action,
compositions, methods, and uses thereof [WO/2010/
120374 (C.-J.L.) and PCT/US2010/001137 (C.-J.L.)]. TNFR2
(TNFRSF1B/CD120b) accession number: NM_130426. A
full list of author contributions is available in the SOM.
Supporting Online Material
Materials and Methods
Figs. S1 to S19
18 October 2010; accepted 28 February 2011
Published online 10 March 2011;
Switch for RPTPs Clustering
and Neuronal Extension
Charlotte H. Coles,1 Yingjie Shen,2 Alan P. Tenney,2,3† Christian Siebold,1†
Geoffrey C. Sutton,1 Weixian Lu,1 John T. Gallagher,4,5 E. Yvonne Jones,1‡
John G. Flanagan,2‡ A. Radu Aricescu1‡
Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous
cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve
regeneration through receptor protein tyrosine phosphatase sigma (RPTPs). Here we report that RPTPs
acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion.
Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that
can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs
induced RPTPs ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate.
RPTPs and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the
extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on
neuronal extension by competing to control the oligomerization of a common receptor.
Type IIa receptor protein tyrosine phos- phatases (RPTPs) are cell surface receptors important for nervous system development,
function, and repair (1–3). Vertebrate family members [RPTPs, leukocyte common antigen-related
(LAR) protein, and RPTPd] and invertebrate
orthologs [e.g., Drosophila LAR (DLAR)] localize to axonal growth cones, regulating neuronal
growth and guidance and participating in excitatory synapse formation and maintenance (1, 4–8).
RPTPs –/– mice exhibit neurological and neuro-endocrine defects (9, 10), as well as increased nerve
regeneration (11–15); RPTPd-deficient mice show
impaired learning and memory (16). RPTPs and
d double-mutant mice have a developmental loss
of motor neurons leading to paralysis (17).
Type IIa RPTP extracellular regions interact with heparan sulfate proteoglycans (HSPGs)
and chondroitin sulfate proteoglycans (CSPGs)
(5, 7, 12, 18). These proteoglycans modulate