High-Concentrations of Fibronectin Fragments Cause Short-Term Catabolic Effects in Cartilage Tissue While Lower Concentrations Cause Continuous Anabolic Effects

doi:10.1006/abbi.1994.1229

Archives of Biochemistry and Biophysics

Volume 311, Issue 2, June 1994, Pages 213-218

https://doi.org/10.1006/abbi.1994.1229 Get rights and content

Abstract

We reported earlier that Fn fragments (Fn-f) added to bovine articular cartilage cultured in serum-free DMEM cause marked elevated release of protease activity within a few days. This results in greatly elevated rates of release of proteoglycan (PG). We have now extended our studies to 4-week cultures of cartilage in the presence of 10% serum. We report here that cartilage cultured with 0.01, 0.1, and 1 μM of an amino terminal 29-kDa Fn-f in 10% serum accelerated the loss of PG from the tissue during the first few days in a concentration-dependent manner. However, beyond this period PG content decreased much more slowly, During this early period, the 29-kDa Fn-f decreased rates of protein and PG synthesis up to 50% in a concentration-dependent fashion. Beyond this period, the synthesis rates began to increase in a mode inversely related to 29-kDa Fn-f concentration, up to 135% of Fn-f free control values. However, during the entire culture period, cartilage cultured with 1 nM 29-kDa Fn-f had higher PG contents and had enhanced rates of protein and PG synthesis. Since 1 nM 29-kDa Fn-f stabilized cartilage against decreases in PG content, we tested its ability to block the activity of higher 29-kDa Fn-f concentrations. Cartilage was preincubated for 7 days with 1 nw 29-kDa Fn-f, and then the culture adjusted to 100 nM 29-kDa Fn-f to cause PG depletion. The preincubated cartilage showed markedly enhanced resistance to PG depletion. Since the protective effect was similar to known properties of IGP-1, the ability of 20 ng/ml IGF-1 to block against the effects of 100 nM Fn-f was tested and shown to be similar in magnitude to that of 1 nM 29-kDa Fn-f. We propose that the initial catabolic effects of higher concentrations of Fn-f, followed by the later anabolic effects, may aid in tissue repair. Also, the continuous anabolic effects of lower concentrations may be involved in tissue homeostasis.

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Cited by (61)

Genetic polymorphisms associated with intervertebral disc degeneration
2013, Spine Journal
Disc degeneration (DD) is a multifaceted chronic process that alters the structure and function of the intervertebral discs and can lead to painful conditions. The pathophysiology of degeneration is not well understood, but previous studies suggest that certain genetic polymorphisms may be important contributing factors leading to an increased risk of DD.
To review the genetic factors in DD with a focus on polymorphisms and their putative role in the pathophysiology of degeneration. Elucidating the genetic components that are associated with degeneration could provide insights into the mechanism of the process. Furthermore, defining these relationships and eventually using them in a clinical setting may allow an identification and early intervention for those who are at a high risk for painful DD.
Literature review.
This literature review focused on the studies concerning genetic polymorphisms and their associations with DD.
Genetic polymorphisms in 20 genes have been analyzed in association with DD, including vitamin D receptor, growth differentiation factor 5 (GDF5), aggrecan, collagen Types I, IX, and XI, fibronectin, hyaluronan and proteoglycan link protein 1 (HAPLN1), thrombospondin, cartilage intermediate layer protein (CILP), asporin, MMP1, 2, and 3, parkinson protein 2, E3 ubiquitin protein ligase (PARK2), proteosome subunit β type 9 (PSMB9), tissue inhibitor of metalloproteinase (TIMP), cyclooxygenase-2 (COX2), and IL1α, IL1β, and IL6. Each genetic polymorphism codes for a protein that has a functional role in the pathogenesis of DD.
There are known associations between several genetic polymorphisms and DD. Of the 20 genes analyzed, polymorphisms in vitamin D receptor, aggrecan, Type IX collagen, asporin, MMP3, IL1, and IL6 show the most promise as functional variants. Genetic studies are crucial for understanding the mechanism of the degeneration. This genetic information could eventually be used as a predictive model for determining a patient’s risk for symptomatic DD.
Type IX collagen interacts with fibronectin providing an important molecular bridge in articular cartilage
2011, Journal of Biological Chemistry
Citation Excerpt :
These FN fragments induce a catabolic response that results in a severe loss of cartilage proteoglycan, an early characteristic of OA, both in vitro and in vivo (54, 55). However, at much lower concentrations, FN fragments have an anabolic effect on cartilage metabolism, such as promoting an increased release of TGF-β and insulin-like growth factor-1 (56, 57). Therefore, FN and FN fragments are implicated as important regulators of normal cartilage homeostasis and cartilage degeneration during disease progression.
Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix. The N-terminal, globular noncollagenous domain (NC4) of the α1(IX) chain protrudes away from the surface of the fibrils into the surrounding matrix and is available for molecular interactions. To define these interactions, we used the NC4 domain in a yeast two-hybrid screen of a human chondrocyte cDNA library. 73% of the interacting clones encoded fibronectin. The interaction was confirmed using in vitro immunoprecipitation and was further characterized by surface plasmon resonance. Using whole and pepsin-derived preparations of type IX collagen, the interaction was shown to be specific for the NC4 domain with no interaction with the triple helical collagenous domains. The interaction was shown to be of high affinity with nanomolar K_d values. Analysis of the fibronectin-interacting clones indicates that the constant domain is the likely site of interaction. Type IX collagen and fibronectin were shown to co-localize in cartilage. This novel interaction between the NC4 domain of type IX collagen and fibronectin may represent an in vivo interaction in cartilage that could contribute to the matrix integrity of the tissue.
Fibronectin fragments mediate matrix metalloproteinase upregulation and cartilage damage through proline rich tyrosine kinase 2, c-src, NF-κB and protein kinase Cδ
2009, Osteoarthritis and Cartilage
Since fibronectin fragments (Fn-fs) enhance cartilage damage through integrins, the objective was to investigate the role of integrin linked kinases, focal adhesion kinase (FAK) and a soluble form of FAK, proline rich tyrosine kinase 2 (Pyk2) and cellular src kinase (c-src) and the transcription factor, nuclear factor κB (NF-κB) in cartilage damage.
Bovine chondrocytes were cultured with various concentrations of three different Fn-fs, an amino-terminal 29 kDa, a gelatin binding 50 kDa and a central 140-kDa Fn-fs, each with progressively weaker cartilage damaging activity, or with native fibronectin (Fn), and lysates probed for activation of the selected kinases. Confocal microscopy was used to visualize intracellular location of activated kinases and NF-κB. Various kinase inhibitors were tested for their effects on Fn-f mediated upregulation of matrix metalloproteinase (MMP)-3 and -13 and cartilage proteoglycan (PG) depletion.
The Fn-fs kinetically enhanced phosphorylation of FAK but did not show a clear dose–response effect. The 29-kDa and 50-kDa Fn-fs enhanced phosphorylation of Pyk2, c-src and NF-κB to a much greater extent than the 140-kDa Fn-f and native Fn and did so as a function of dose. The 29-kDa Fn-f enhanced the phosphorylation of nuclear Pyk2 as compared with no treatment or native Fn. Inhibitors of Pyk2, c-src, NF-κB and protein kinase Cδ (PKCδ) decreased MMP upregulation and decreased Fn-f mediated damage to cartilage.
These studies enhance our knowledge of crucial factors in Fn-f mediated signaling in MMP upregulation and cartilage damage and because of the potential physiologic relevance of Fn-fs, provide a better knowledge of cartilage degeneration in general.
The cartilage chondrolytic mechanism of fibronectin fragments involves MAP kinases: comparison of three fragments and native fibronectin
2008, Osteoarthritis and Cartilage
Citation Excerpt :
We have reported that fibronectin fragments (Fn-fs) have cartilage chondrolytic activities1, elevate matrix metalloproteinase (MMP) expression2–4, suppress proteoglycan (PG) synthesis in chondrocytes and cartilage5–7 and enhance rates of PG loss from cartilage tissue in explant cultures1–3,5–7.
To define the role of mitogen activated protein (MAP) kinases in fibronectin fragment (Fn-f) mediated matrix metalloproteinase (MMP) upregulation and damage to bovine cartilage and to compare activities of three Fn-fs with native fibronectin (Fn), which is inactive in terms of cartilage damage.
Bovine chondrocytes were cultured with three Fn-fs, an amino-terminal 29-kDa, a gelatin-binding 50-kDa and a central 140-kDa Fn-f or native Fn at concentrations from 0.01 to 1 μM, concentrations lower than those found in osteoarthritis synovial fluids. Lysates were probed for activation of MAP kinases, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and stress activated protein kinase/c-jun N-terminal kinase (SAPK/JNK). Confocal fluorescent microscopy was used to visualize movement of activated kinases. Kinase inhibitors were tested for their abilities to block Fn-f mediated protein upregulation of MMP-3 and MMP-13 and Fn-f induced depletion of cartilage proteoglycan (PG) from cultured explants.
The 29-kDa, the most potent Fn-f in terms of cartilage damage, enhanced phosphorylation of ERK1/2, p38 and JNK1/2 within a 1-h incubation while the 50 and 140-kDa Fn-fs required up to 4 h for maximal activity and native Fn was only minimally active toward p38 and JNK, but did strongly activate ERK1/2. The activated kinases displayed a distribution toward the nuclear membrane and within the nucleus. MAP kinase inhibitors markedly decreased Fn-f mediated upregulation of MMP-3 or MMP-13 and Fn-f mediated cartilage PG depletion.
These results suggest that Fn-fs upregulate MMP-3 and MMP-13 in bovine chondrocytes through MAP kinases and that kinase inhibitors afford protection against this degenerative pathway.
Mixtures of glucosamine and chondroitin sulfate reverse fibronectin fragment mediated damage to cartilage more effectively than either agent alone
2006, Osteoarthritis and Cartilage
Citation Excerpt :
This latter culture system allowed us to measure effects of agents on homeostasis closer to steady state conditions than serum-free and thus, to measure the summation of anabolic and catabolic events. We have shown that in these long-term 21–28 day cultures, that cell viability as measured by DNA content does not decrease by more than 20%, an amount not statistically different than untreated cartilage54. Bovine cartilage was cultured in 10% serum/DMEM with the single agents or with a mixture and with or without 100 nM Fn-f for periods up to 21 or 28 days.
To test the effectiveness of glucosamine (GluNH₂)-HCl, chondroitin sulfate (CS) and mixtures in protecting cartilage exposed to fibronectin fragments (Fn-fs), an exposure known to enhance catabolic cytokines and matrix metalloproteinases (MMPs).
Pharmacologic formulations of GluNH₂ (FCHG49^®) and CS (TRH122^®) (Nutramax Laboratories, Inc.) were added at 1, 10 or 100 μg/ml singly or in mixtures to bovine cartilage cultures in serum or serum-free conditions with or without Fn-f. Proteoglycan (PG) release into media and remaining cartilage PG content were measured by dye binding analysis and effects on PG synthesis by assays of 35-sulfate incorporation. Effects on MMP-3 and -13 expression were measured by Western blotting of conditioned media.
In serum-free conditions, the agents singly or as mixtures did not block Fn-f mediated matrix degradation. In serum, single agents were weakly effective at 100 μg/ml, while the mixture of each agent at 0.1 μg/ml decreased PG loss by about 50% by day 7 and at 1 μg/ml restored nearly 50% of the PG after 7 days in Fn-f pretreated cartilage. However, both agents singly and as mixtures at 0.1–100 μg/ml decreased MMP release. In serum, the single agents at 1–10 μg/ml weakly reversed Fn-f mediated PG synthesis suppression, while the mixtures were 100% effective at 1 μg/ml.
GluNH₂ and CS act synergistically in reversing damage and promoting repair at concentrations found in plasma after oral ingestion of these agents. Reversal of PG synthesis suppression correlates more with these activities than suppression of MMP-3 or -13 expression.
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Regular ArticleHigh-Concentrations of Fibronectin Fragments Cause Short-Term Catabolic Effects in Cartilage Tissue While Lower Concentrations Cause Continuous Anabolic Effects

Abstract

Regular Article
High-Concentrations of Fibronectin Fragments Cause Short-Term Catabolic Effects in Cartilage Tissue While Lower Concentrations Cause Continuous Anabolic Effects