Transforming growth factor-β in calf articular cartilage organ cultures: Synthesis and distribution
Abstract
Transforming growth factor β1 (TGF-β1) has been shown to play a prominent role in controlling proteoglycan synthesis and breakdown as measured following addition to organ cultures of calf articular cartilage (Morales, T. I., and Roberts, A. B., J. Biol. Chem., 263, 12,828–12,831, 1988). In this study, we compare two closely related TGF-β isoforms, TGF-β1 and TGF-β2, both by assessing the effects of exogenous peptide as well as by analyzing the biosynthesis and total amount of these two isoforms in cartilage explants. Added exogenously, TGF-β1 and TGF-β2 induce a comparable increase in proteoglycan synthesis over basal controls with saturation at ~5 ng/ml. Synthesis of TGF-β by basal calf cartilage cultures is demonstrated by (i) immunolocalization of intracellular TGF-β, (ii) Northern blot analysis of steady-state mRNA levels, and (iii) immunoprecipitation of metabolically labeled TGF-β from tissue extracts and conditioned culture medium. The net amount of extractable TGF-β1 and TGF-β2 in the basal cartilage cultures was assessed by a functional assay involving inhibition of proliferation of CCL-64 mink lung epithelial cells and by sandwich enzyme-linked immunosorbent assay. The predominant isoform was TGF-β1 (60–85%) and the total TGF-β1 + TGF-β2 was in excess of the amount required for maximal activation of proteoglycan synthesis. The level of both isoforms was maintained relatively constant between Days 2 and 7 of culture despite a sharp (~ two to fourfold) drop in proteoglycan synthesis. This suggests that cartilage contains a large pool of TGF-β which is not readily accessible to the chondrocyte. We propose that much of the polypeptide is sequestered in the matrix awaiting release upon demand.
References (38)
- T.I. Morales et al.
J. Biol. Chem
(1988) - T.I. Morales
Arch. Biochem. Biophys
(1991) - S.J. Kim et al.
J. Biol. Chem
(1989) - S.J. Kim et al.
J. Biol. Chem
(1989) - V.C. Hascall et al.
Arch. Biochem. Biophys
(1983) - T.I. Morales et al.
J. Biol. Chem
(1984) - J.H. Kimura et al.
J. Biol. Chem
(1981) - J.F. Woessner
Arch. Biochem. Biophys
(1961) - L.R. Ellingsworth et al.
J. Biol. Chem
(1986) - L.M. Wakefield et al.
J. Biol. Chem
(1988)
ISI Atlas Sci. Biochem
Nature
Cell Reg
Mol. Cell. Biol
Cell Reg
Connect. Tissue Res
J. Cell. Biol
Cited by (116)
Growth factors that drive aggrecan synthesis in healthy articular cartilage. Role for transforming growth factor-β?
2024, Osteoarthritis and Cartilage OpenArticular cartilage makes smooth movement possible and destruction of this tissue leads to loss of joint function. An important biomolecule that determines this function is the large aggregating proteoglycan of cartilage, aggrecan. Aggrecan has a relatively short half-life in cartilage and therefore continuous production of this molecule is essential.
In this narrative review we discuss what is the role of growth factors in driving the synthesis of aggrecan in articular cartilage. A literature search has been done using the search items; cartilage, aggrecan, explant, Transforming Growth factor-β (TGF-β), Insulin-like Growth Factor (IGF), Bone Morphogenetic Protein (BMP) and the generic term “growth factors”. Focus has been on studies using healthy cartilage and models of cartilage regeneration have been excluded.
In healthy adult articular cartilage IGF is the main factor that drives aggrecan synthesis and maintains adequate levels of production. BMP's and TGF-β have a very limited role but appear to be more important during chondrogenesis and cartilage development. The major role of TGF-β is not stimulation of aggrecan synthesis but maintenance of the differentiated articular cartilage chondrocyte phenotype.
TGF-β is a factor that is generally considered as an important factor in stimulating aggrecan synthesis in cartilage but its role in this might be very restrained in healthy, adult articular cartilage.
Computational and experimental characterizations of the spatiotemporal activity and functional role of TGF-β in the synovial joint
2023, Journal of BiomechanicsTGF-β is a prominent anabolic signaling molecule associated with synovial joint health. Recent work has uncovered mechanochemical mechanisms that activate the latent form of TGF-β (LTGF-β) in the synovial joint—synovial fluid (SF) shearing or cartilage compression—pointing to mechanobiological phenomena, whereby enhanced TGF-β activity occurs during joint stimulation. Here, we implement computational and experimental models to better understand the role of mechanochemical-activated TGF-β (aTGF-β) in regulating the functional biosynthetic activities of synovial joint tissues. Reaction-diffusion models describe the pronounced role of extracellular chemical reactions—load-induced activation, reversible ECM-binding, and cell-mediated internalization—in modulating the spatiotemporal distribution of aTGF-β in joint tissues. Of note, aTGF-β from SF shearing predominantly acts on cells in peripheral tissue regions (superficial zone [SZ] chondrocytes and synoviocytes) and aTGF-β from cartilage compression acts on chondrocytes through all cartilage layers. Further, ECM reversible binding sites in cartilage act to modulate the temporal delivery of aTGF-β to cells, creating a dynamic where short durations of joint activity give rise to extended periods of aTGF-β exposure at moderated doses. Ex vivo tissue models were subsequently utilized to characterize the influence of physiologic aTGF-β activity regimens in regulating functional biosynthetic activities. Physiologic exposure regimens of aTGF-β in SF induce strong 4-fold to 9-fold enhancements in the secretion rate of the synovial biolubricant, PRG4, from SZ cartilage and synovium explants. Further, aTGF-β inhibition in cartilage over 1-month culture leads to a pronounced loss of GAG content (30–35% decrease) and tissue softening (60–65% EY reduction). Overall, this work advances a novel perspective on the regulation of TGF-β in the synovial joint and its role in maintaining synovial joint health.
A chemo-mechano-biological modeling framework for cartilage evolving in health, disease, injury, and treatment
2023, Computer Methods and Programs in BiomedicineOsteoarthritis (OA) is a pervasive and debilitating disease, wherein degeneration of cartilage features prominently. Despite extensive research, we do not yet understand the cause or progression of OA. Studies show biochemical, mechanical, and biological factors affect cartilage health. Mechanical loads influence synthesis of biochemical constituents which build and/or break down cartilage, and which in turn affect mechanical loads. OA-associated biochemical profiles activate cellular activity that disrupts homeostasis. To understand the complex interplay among mechanical stimuli, biochemical signaling, and cartilage function requires integrating vast research on experimental mechanics and mechanobiology—a task approachable only with computational models. At present, mechanical models of cartilage generally lack chemo-biological effects, and biochemical models lack coupled mechanics, let alone interactions over time.
We establish a first-of-its kind virtual cartilage: a modeling framework that considers time-dependent, chemo-mechano-biologically induced turnover of key constituents resulting from biochemical, mechanical, and/or biological activity. We include the “minimally essential” yet complex chemical and mechanobiological mechanisms. Our 3-D framework integrates a constitutive model for the mechanics of cartilage with a novel model of homeostatic adaptation by chondrocytes to pathological mechanical stimuli, and a new application of anisotropic growth (loss) to simulate degradation clinically observed as cartilage thinning.
Using a single set of representative parameters, our simulations of immobilizing and overloading successfully captured loss of cartilage quantified experimentally. Simulations of immobilizing, overloading, and injuring cartilage predicted dose-dependent recovery of cartilage when treated with suramin, a proposed therapeutic for OA. The modeling framework prompted us to add growth factors to the suramin treatment, which predicted even better recovery.
Our flexible framework is a first step toward computational investigations of how cartilage and chondrocytes mechanically and biochemically evolve in degeneration of OA and respond to pharmacological therapies. Our framework will enable future studies to link physical activity and resulting mechanical stimuli to progression of OA and loss of cartilage function, facilitating new fundamental understanding of the complex progression of OA and elucidating new perspectives on causes, treatments, and possible preventions.
Inhibition of transforming growth factor-β in osteoarthritis. Discrepancy with reduced TGFβ signaling in normal joints
2022, Osteoarthritis and Cartilage OpenTransforming growth factor-β (TGFβ) is a pleiotropic cytokine that is central in the regulation of joint health and disease. Inhibition of TGFβ activity/signaling in experimental osteoarthritis (OA) has been performed to modulate OA severity and progression. In this narrative review we discuss the potential reasons for the variable results of TGFβ inhibition in these models.
A literature study was performed using the search terms; experimental osteoarthritis and TGFβ. Papers were selected that describe the effect TGFβ activity/signaling inhibition on experimental OA. Based on the selected papers a narrative review has been written about the potential therapeutic role of TGFβ inhibition in OA and potential causes for its variable effects are discussed.
Inhibition of TGFβ activity in experimental models of OA does not result in either straightforward protection or deleterious effects. More than half of the studies (13/19), but not all, report that inhibition of TGFβ in experimental OA reduces OA severity. This is in contrast with the protective role of TGFβ in healthy joints.
The effect of TGFβ inhibition on joint damage in experimental OA is variable. Most likely this is a consequence of the changing function of TGFβ in normal and OA joints. As a result, the overall outcome of TGFβ modulation in OA will be unpredictable. To develop OA therapies based on modulation of TGFβ activity specific protective and damaging signaling routes should be identified and tools developed to block the damaging ones.
Nanofiber-based transforming growth factor-β3 release induces fibrochondrogenic differentiation of stem cells
2019, Acta BiomaterialiaFibrocartilage is typically found in regions subject to complex, multi-axial loads and plays a critical role in musculoskeletal function. Mesenchymal stem cell (MSC)-mediated fibrocartilage regeneration may be guided by administration of appropriate chemical and/or physical cues, such as by culturing cells on polymer nanofibers in the presence of the chondrogenic growth factor TGF-β3. However, targeted delivery and maintenance of effective local factor concentrations remain challenges for implementation of growth factor-based regeneration strategies in clinical settings. Thus, the objective of this study was to develop and optimize the bioactivity of a biomimetic nanofiber scaffold system that enables localized delivery of TGF-β3. To this end, we fabricated TGF-β3-releasing nanofiber meshes that provide sustained growth factor delivery and demonstrated their potential for guiding synovium-derived stem cell (SDSC)-mediated fibrocartilage regeneration. TGF-β3 delivery enhanced cell proliferation and synthesis of relevant fibrocartilaginous matrix in a dose-dependent manner. By designing a scaffold that eliminates the need for exogenous or systemic growth factor administration and demonstrating that fibrochondrogenesis requires a lower growth factor dose compared to previously reported, this study represents a critical step towards developing a clinical solution for regeneration of fibrocartilaginous tissues.
Fibrocartilage is a tissue that plays a critical role throughout the musculoskeletal system. However, due to its limited self-healing capacity, there is a significant unmet clinical need for more effective approaches for fibrocartilage regeneration. We have developed a nanofiber-based scaffold that provides both the biomimetic physical cues, as well as localized delivery of the chemical factors needed to guide stem cell-mediated fibrocartilage formation. Specifically, methods for fabricating TGF-β3-releasing nanofibers were optimized, and scaffold-mediated TGF-β3 delivery enhanced cell proliferation and synthesis of fibrocartilaginous matrix, demonstrating for the first time, the potential for nanofiber-based TGF-β3 delivery to guide stem cell-mediated fibrocartilage regeneration. This nanoscale delivery platform represents an exciting new strategy for fibrocartilage regeneration.
Effect of TGF-β1 on water retention properties of healthy and osteoarthritic chondrocytes
2018, Materials Today: ProceedingsOsteoarthritis (OA) is a progressive degenerative disease, which includes reduction of cartilage thickness between two bones in a joint, causing painful bone-to-bone contact. OA affects over 8 million people in the UK alone, and as the primary causes are unknown, available treatments only reduce the symptoms instead of providing a cure. This project focused on utilizing TGF-β1, a cytokine found in elevated amounts in healthy cartilage when compared to degraded cartilage, in order to observe the effects of the growth factor on both healthy and osteoarthritic chondrocytes. The healthy and the osteoarthritic chondrocytes were cultured in two different media (DMEM with and without TGF- β1) before utilizing the SpectraMax M2/M2e plate reader to observe and analyze the effect of TGF-β1 on water retention properties of cells. This has been achieved by quantifying the GAG content using DMMB dye. Results showed that although TGF-β1 displayed an increase in glycosaminoglycan synthesis, the statistical increase was not vast enough for the alternative hypothesis to be accepted; further experimentation with TGF-β1, alongside other cytokines within the growth factor family is needed to perceive the true influence of the growth factor on uncured degenerative diseases. It was concluded that both the healthy and osteoarthritic cells treated with TGF-β1 absorbed considerably more DMMB in comparison to the cells, suggesting that TGF-β1 indeed works to aid in water retention.