An Elisa Plate Based Assay for Hyaluronan Using Biotinylated Proteoglycan G1 Domain (HA-Binding Region)

Fosang, Amanda J.; Hey, Nina J.; Carney, Stephen; Hardingham, Timothy E.

doi:10.1016/s0934-8832(11)80186-1

Cited by 117 publications

(62 citation statements)

References 24 publications

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“…Hyaluronan (HA)-dependent matrix assembly was verified by treatment for 1 h with 6 units ml Ϫ1 Streptomyces hyalurolyticus hyaluronidase (Sigma). HA Assay-An enzyme-linked immunosorbance-based assay was used to measure concentrations of HA released into the culture media 24 h after mechanical strain application (31). The assay is based upon the competition between HA absorbed on to the plate and HA free in solution for binding to biotinylated cartilage proteoglycan binding region (G1 domain) (32).…”

Section: Methodsmentioning

confidence: 99%

A Specific Mechanomodulatory Role for p38 MAPK in Embryonic Joint Articular Surface Cell MEK-ERK Pathway Regulation

Lewthwaite

Bastow

Lamb

et al. 2006

Journal of Biological Chemistry

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Mechanisms regulating cell behavior and extracellular matrix composition in response to mechanical stimuli remain unresolved. Our previous studies have established that the MEK-ERK cascade plays a specific role in the mechano-dependent joint formation process by promoting the assembly of pericellular matrices reliant upon hyaluronan (HA) for their integrity. Here we demonstrate: (i) novel cross-talk between p38 MAPK and MEK-ERK signaling pathways that is specific for mechanical stimuli and (ii) a role for p38 MAPK in facilitating HA production by cells derived from the articular surface of embryonic chick tibiotarsal joints. We find that p38 MAPK blockade restricts pericellular assembly of HA-rich matrices and reduces basal as well as mechanical strain-induced release of HA. p38 MAPK blockers potentiated early strain-induced increases but restricted sustained increases in MEK/ERK phosphorylation at later times; c-Fos hyperphosphorylation at threonine 325 was found to parallel this p38 MAPK-mediated modulation of ERK activation. In contrast, p38 MAPK inhibitors had no detectable effect on the ERK activation induced by fibroblast growth factor 2 or pervanadate, a phosphatase inhibitor, and MEK inhibitors did not influence p38 MAPK phosphorylation, confirming both the specificity and unidirectionality of p38 MAPK-ERK cross-talk. Immunochemical and immunoblotting studies revealed constitutive p38 MAPK activation in cells at, or derived from, developing articular joint surfaces. Unlike the MEK-ERK pathway, however, p38 MAPK was not further stimulated by mechanical stimulation in vitro. Thus, p38 MAPK specifically facilitates ERK activation and downstream signaling in response to mechanical stimuli. These results suggest that constitutively active p38 MAPK serves an essential, permissive role in mechanically induced changes in ERK activation and in the accumulation of HA-rich extracellular matrices that serve a key role in joint development.Mitogen-activated protein kinases (MAPKs) 3 are ubiquitous serine/ threonine kinases that are activated by diverse extracellular stimuli, including growth factors, cytokines, and physiological mechanical signals (1-3). MAPKs are essential for transducing signals from the cell surface that regulate diverse cellular behaviors, such as those coordinating development, proliferation, and differentiation. Much recent emphasis has been placed on members of three well characterized mammalian MAPK families, comprising extracellular signal-regulated kinases (ERKs), p38 MAPKs, and c-Jun N-terminal kinases (JNKs) (4 -6). Until relatively recently it was considered that these families were preferentially activated by certain types of signal: the ERK family by growth factors and the p38 MAPK and JNK families by cellular stress. It is now becoming increasingly clear, however, that the various MAPK pathways can also be co-activated by a single stimulus. The precise interaction between members of these distinct MAPK families and how they cooperate to control cell behavior are, however, ill-define...

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Section: Methodsmentioning

confidence: 99%

A Specific Mechanomodulatory Role for p38 MAPK in Embryonic Joint Articular Surface Cell MEK-ERK Pathway Regulation

Lewthwaite

Bastow

Lamb

et al. 2006

Journal of Biological Chemistry

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“…The HA present in ADAMTS digestion supernatants or cartilage extracts was measured using a commercial HA enzyme-linked immunosorbent assay (ELISA) kit (product no. K-1200; Echelon Biosciences, Salt Lake City, UT), wherein samples are analyzed in a competition assay in a format similar to that described elsewhere (23). To eliminate competition by endogenous HA binding proteins, samples were reduced and alkylated or digested with proteinase K as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS‐4 (aggrecanase 1) or ADAMTS‐5 (aggrecanase 2)

Chockalingam¹,

Zeng²,

Morris³

et al. 2004

Arthritis & Rheumatism

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Objective. To determine whether aggrecanase (ADAMTS) activities in articular cartilage can directly lead to the release of hyaluronan (HA) and hyaladherins (aggrecan G1 domain and link proteins), as may occur ex vivo during stimulation of cartilage explants with interleukin-1 (IL-1) or retinoic acid or in vivo in synovial joints during aging and joint pathology.Methods. Bovine articular cartilage discs (live or freeze-killed) were cultured in the presence of IL-1 or were incubated in digestion buffer containing recombinant human ADAMTS-4 (rHuADAMTS-4; aggrecanase 1) or rHuADAMTS-5 (aggrecanase 2). Culture media, digestion supernatants, and tissue extracts were assayed for sulfated glycosaminoglycan (sGAG) content and analyzed by Western blotting to detect aggrecanasegenerated G1 domain (using neoepitope monoclonal antibody AGG-C1/anti-NITEGE 373 ) and link proteins (using monoclonal antibody 8-A-4), as well as by quantitative enzyme-linked immunosorbent assays to detect aggrecanase-generated G1 domain (G1-NITEGE 373 ) and HA.Results. IL-1 treatment of live cartilage explants induced a time-dependent release of sGAG, aggrecanase-generated G1 domain (G1-NITEGE 373 ), and HA into the culture media. Exposure of live or freeze-killed articular cartilage discs to rHuADAMTS-4 or rHuADAMTS-5 resulted in a dose-and timedependent release of sGAG and hyaluronan from the tissue, accompanied by a concomitant release of functionally intact hyaladherins (aggrecan G1-NITEGE 373 and link proteins).Conclusion. Coincident with aggrecanolysis, aggrecanase activities in articular cartilage may actuate the release of HA and associated hyaladherins, thereby further compromising the integrity of the cartilage matrix during degenerative joint diseases such as osteoarthritis.

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“…Lp, G1 and biotinylated G1 (b-G1) were prepared from porcine laryngeal cartilage as described before [4,22]; Lp was stored in 4 M guanidine-HCl, 50 mM Na-acetate, 1 mM Na P -EDTA, pH 5.8, due to its low solubility in water. Both G1 and b-G1, following biotinylation in the presence of HA, were puri¢ed by gel ¢ltration under dissociative conditions (i.e.…”

Section: Methodsmentioning

confidence: 99%

TSG‐6 interacts with hyaluronan and aggrecan in a pH‐dependent manner via a common functional element: implications for its regulation in inflamed cartilage

et al. 1998

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Cartilage matrix is stabilised by the interactions of proteins with hyaluronan (HA). We compare the pH dependences of HA binding by aggrecan, link protein and TSG-6. Aggrecan and link protein exhibit maximal binding across a wide pH range (6.0^8.0). TSG-6, a protein that is only produced during inflammation, binds maximally at about pH 6.0 but shows a dramatic loss of function with increasing pH. TSG-6 also interacts with aggrecan, with a similar pH dependence, and this can be inhibited by HA. Thus, a common binding surface on TSG-6 may be involved in HA and aggrecan binding. We propose that TSG-6 is involved in matrix dissociation and that this is regulated by pH gradients in cartilage.z 1998 Federation of European Biochemical Societies.

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An Elisa Plate Based Assay for Hyaluronan Using Biotinylated Proteoglycan G1 Domain (HA-Binding Region)

Cited by 117 publications

References 24 publications

A Specific Mechanomodulatory Role for p38 MAPK in Embryonic Joint Articular Surface Cell MEK-ERK Pathway Regulation

A Specific Mechanomodulatory Role for p38 MAPK in Embryonic Joint Articular Surface Cell MEK-ERK Pathway Regulation

Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS‐4 (aggrecanase 1) or ADAMTS‐5 (aggrecanase 2)

TSG‐6 interacts with hyaluronan and aggrecan in a pH‐dependent manner via a common functional element: implications for its regulation in inflamed cartilage

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