The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan

Flory, Jennifer J. Embry; Fosang, Amanda J.; Knudson, Warren

doi:10.1002/art.21623

Cited by 26 publications

(31 citation statements)

References 50 publications

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“…However, the lack of crossreactivity between antibodies to C1,2C, Coll2-1, and Coll2-1NO 2 and denatured collagen molecules (13,19) is not consistent with this hypothesis. A second, more probable, explanation is that intracellular staining reflects cellular uptake of degraded collagen molecules, as previously shown for hyaluronan and aggrecan fragments (39,40). If this is indeed the case, labeling of healthy chondrocytes not only in WT mice, but also in biglycan/fibromodulin double-deficient mice, reflects physiologic type II collagen turnover.…”

Section: Discussionmentioning

confidence: 77%

The chemical biomarkers C2C, Coll2‐1, and Coll2‐1NO₂ provide complementary information on type II collagen catabolism in healthy and osteoarthritic mice

Ameye¹,

Deberg²,

Oliveira³

et al. 2007

Arthritis & Rheumatism

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Objective. Compared with wild-type (WT) mice, biglycan/fibromodulin double-deficient mice develop severe knee osteoarthritis. We undertook this study to compare type II collagen catabolism in the 2 genotypes and to compare the usefulness of 3 biomarkers of collagen degradation (C2C [also known as Col2-3/4C long mono ] as well as the peptide Coll2-1 and its nitrated form, Coll2-1NO 2 ) for evaluating collagen catabolism in vivo.Methods. In 15 WT mice and 15 biglycan/ fibromodulin double-deficient mice, we determined serum levels of C2C at ages 66 and 141 days, and we determined serum levels of Coll2-1 and Coll2-1NO 2 at ages 49, 81, 95, and 141 days. Expression of the biomarkers in knee sections was examined using immunohistochemistry.Results. The mean concentrations of C2C and Coll2-1 were higher in biglycan/fibromodulin doubledeficient mice at all time points. For C2C and Coll2-1, the ratio of the serum concentration in biglycan/ fibromodulin double-deficient mice to that in WT mice (the double-deficient:WT ratio) was constant over time and was ϳ1.63 and ϳ1.15, respectively. In contrast, the double-deficient:WT ratio for Coll2-1NO 2 varied and, depending on age, was >1 or <1. No significant correlation was found between the expression of the different biomarkers, except for a weak, negative correlation between Coll2-1NO 2 and C2C. In both genotypes, antibodies to each biomarker labeled some fibroblasts in the tendons and menisci as well as chondrocytes above the tidemark in articular cartilage. Growth plates were unstained. For each biomarker, extracellular staining was limited to fibrocartilage areas in the tendons and menisci in all mice and was limited to some focal lesions of the cartilage in biglycan/fibromodulin doubledeficient mice.Conclusion. The different double-deficient:WT ratios observed with C2C, Coll2-1, and Coll2-1NO 2 in the absence of any correlation between the expression of the 3 biomarkers indicate that these biomarkers give complementary, rather than redundant, information about in vivo type II collagen catabolism.Osteoarthritis (OA) is one of the leading causes of pain and disability in the elderly. Its high prevalence and its moderate-to-severe impact on daily life pose a significant public health problem (1). Despite intensive research, a cure remains elusive for this disease with important socioeconomic consequences. The intrinsic difficulty in finding a cure is compounded by the low sensitivity of diagnostic and monitoring tools. For example, the destruction of articular cartilage is an important feature of OA, and radiographic change in joint space width (JSW) is widely recognized as the gold standard end point for measuring destruction of articular cartilage and OA progression. However, this end point is an indirect measure of cartilage integrity, since cartilage is invisible on radiographs and its integrity must be inferred from the spacing between bones. Furthermore, change in JSW has a low signal-to-noise ratio; annual changes are only 0.1-0.2 mm in OA patients (2), close to the p...

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Section: Discussionmentioning

confidence: 77%

The chemical biomarkers C2C, Coll2‐1, and Coll2‐1NO₂ provide complementary information on type II collagen catabolism in healthy and osteoarthritic mice

Ameye¹,

Deberg²,

Oliveira³

et al. 2007

Arthritis & Rheumatism

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“…It would seem that aggrecan fragments generated in vitro, by either aggrecanases or MMPs, are internalized, which may be the final pathway for complete catabolism of aggrecan. 23 In contrast, type VI collagen showed a two-layer distribution around the cells in all groups where the spatial distribution of the inner layer changed little over time. Taken together, COMP and aggrecan showed a rapid turnover than collagen in chondrocyte/agarose constructs.…”

Section: Discussionmentioning

confidence: 81%

Effects of Serum and Compressive Loading on the Cartilage Matrix Synthesis and Spatiotemporal Deposition Around Chondrocytes in 3D Culture

DeLassus

Patra

Liao

et al. 2013

Tissue Engineering Part A

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The aim of this study was to investigate the effects of serum and compressive dynamic loading on the cartilaginous matrix spatiotemporal distribution around chondrocytes in vitro. Murine chondrocytes suspended in agarose were cultured in serum-free media or in varying concentrations of serum with or without compressive dynamic loading. Gene expression was assayed by quantitative polymerase chain reaction. Immunohistochemistry was performed for type II collagen and type VI collagen, aggrecan, or cartilage oligomeric matrix protein (COMP) to study the effect of serum and dynamic loading on the spatiotemporal distribution of cartilage matrix components. Chondrocytes in serum-free culture exhibited negligible differences in type II collagen, aggrecan, and COMP mRNA expression levels over 15 days of cultivation. However, higher serum concentrations decreased matrix gene expression. Expression of the matrix metalloproteinases (MMP)-3 and MMP-13 mRNA increased over time in serum-free or reduced serum levels, but was significantly suppressed in 10% fetal bovine serum (FBS). Compressive loading significantly stimulated MMP-3 expression on days 7 and 15. Immunohistochemical analysis demonstrated that maximum pericellular matrix deposition was achieved in 10% FBS culture in the absence of compressive loading. The pericellular distribution of type II and VI collagens, aggrecan, and COMP proteins tended to be more co-localized in the pericellular region from day 9 to day 21; compressive loading helped promote this co-localization of matrix proteins. The results of this study suggest that the quantity, quality, and spatial distribution of cartilaginous matrix can be altered by serum concentrations and compressive loading.

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“…Although previous studies have documented that G1-ITEGE can be internalized by chondrocytes (19,33), these earlier works were primarily based on immunohistological/cytological analyses. One goal of this study was to demonstrate directly the retention and internalization of the 68 -72-kDa G1-ITEGE by way of Western blotting.…”

Section: Discussionmentioning

confidence: 99%

“…Aliquots of each fraction were analyzed by Western blot analysis. Alternatively, cells before and after hyaluronidase treatment were fixed, permeabilized, and then analyzed by immunofluorescence (21,33). Briefly, after washing in PBS the chondrocytes were incubated with rabbit anti-ITEGE 373 (0.5 g/ml) antibodies overnight at 4°C as described (19,21).…”

Section: Detection Of Cell Surface and Intracellular Aggrecan G1-itegmentioning

confidence: 99%

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Internalization of Aggrecan G1 Domain Neoepitope ITEGE in Chondrocytes Requires CD44

Ariyoshi

Knudson

Luo

et al. 2010

Journal of Biological Chemistry

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Degradation of the cartilage proteoglycan aggrecan is one of the earliest events that occurs in association with osteoarthritis. Little is known concerning the fate of the residual N-terminal G1 domains of cleaved aggrecan; domains that remain bound to hyaluronan. In this study, 68 -72-kDa bands representative of aggrecan G1 domains containing ITEGE 373 neoepitope were detected within a hyaluronidase-sensitive pool at the cell surface of bovine articular chondrocytes and within a hyaluronidase-insensitive, intracellular pool. To determine the mechanisms that contribute to this distribution, CD44 expression was knocked down by siRNA or function by CD44-DN. Both approaches prevented the retention and internalization of G1-ITEGE. Inhibition of CD44 transit into lipid rafts blocked the endocytosis of G1-ITEGE but not the retention at the cell surface. Chondrocytes derived from CD44 null mice also exhibited limited potential for retention and internalization of G1-VTEGE. The consequence of a lack of chondrocyte-mediated endocytosis of these domains in cartilage of the CD44 null mice was the accumulation of the degradation fragments within the tissue. Additionally, chondrocytes or fibroblasts derived from CD44 null mice exhibited little capacity for retention and internalization of exogenous G1-ITEGE derived from bovine cartilage explants. Bovine or wild type mouse fibroblasts were able to bind and internalize bovine-derived G1-ITEGE. Although several pathways are available for the clearance of these domains, CD44-mediated cellular internalization is the most prominent.In cartilage the maintenance of a highly hydrated extracellular matrix is essential for the biomechanical function of the tissue. The primary extracellular macromolecule responsible for this hydration is the proteoglycan aggrecan. Aggrecan is organized as a multiunit aggregate composed of aggrecan monomers bound to a core filament of hyaluronan (HA) 2 and stabilized by association of link proteins (1, 2). The HA/aggrecan/link protein complex is retained at the chondrocyte cell surface either via HA that maintains connections with an HA synthase or HA bound to CD44 (3, 4). All three of these matrix components constantly undergo turnover in healthy cartilage. When studied by precursor radiolabeling, it was found that the biosynthetic rates for HA and aggrecan were remarkably similar (5, 6). Given that the total overall content of HA and aggrecan remains constant led to the conclusion that the two macromolecules must be turned over at the same rate. Subsequent pulse-chase studies confirmed this suggestion (5, 6). Moreover, even when culture conditions were altered such that tissue depletion was enhanced, the elevated rates of turnover for aggrecan and HA remained coordinated (6). The observations imply that the various extracellular processing pathways responsible for the turnover of aggrecan and HA must interact to achieve this level of coordination.One of the early events associated with osteoarthritis is the pronounced loss of aggrecan from the cartilag...

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The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan

Cited by 26 publications

References 50 publications

The chemical biomarkers C2C, Coll2‐1, and Coll2‐1NO₂ provide complementary information on type II collagen catabolism in healthy and osteoarthritic mice

The chemical biomarkers C2C, Coll2‐1, and Coll2‐1NO₂ provide complementary information on type II collagen catabolism in healthy and osteoarthritic mice

Effects of Serum and Compressive Loading on the Cartilage Matrix Synthesis and Spatiotemporal Deposition Around Chondrocytes in 3D Culture

Internalization of Aggrecan G1 Domain Neoepitope ITEGE in Chondrocytes Requires CD44

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The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan

Cited by 26 publications

References 50 publications

The chemical biomarkers C2C, Coll2‐1, and Coll2‐1NO2 provide complementary information on type II collagen catabolism in healthy and osteoarthritic mice

The chemical biomarkers C2C, Coll2‐1, and Coll2‐1NO2 provide complementary information on type II collagen catabolism in healthy and osteoarthritic mice

Effects of Serum and Compressive Loading on the Cartilage Matrix Synthesis and Spatiotemporal Deposition Around Chondrocytes in 3D Culture

Internalization of Aggrecan G1 Domain Neoepitope ITEGE in Chondrocytes Requires CD44

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The chemical biomarkers C2C, Coll2‐1, and Coll2‐1NO₂ provide complementary information on type II collagen catabolism in healthy and osteoarthritic mice

The chemical biomarkers C2C, Coll2‐1, and Coll2‐1NO₂ provide complementary information on type II collagen catabolism in healthy and osteoarthritic mice