Dongyao Yan

A current review of molecular mechanisms regarding osteoarthritis and pain

Osteoarthritis afflicts millions of individuals across the world resulting in impaired quality of life and increased health costs. To understand this disease, physicians have been studying risk factors, such as genetic predisposition, aging, obesity, and joint malalignment; however have been unable to conclusively determine the direct etiology. Current treatment options are short-term or ineffective and fail to address pathophysiological and biochemical mechanisms involved with cartilage degeneration and the induction of pain in arthritic joints. OA pain involves a complex integration of sensory, affective, and cognitive processes that integrate a variety of abnormal cellular mechanisms at both peripheral and central (spinal and supraspinal) levels of the nervous system Through studies examined by investigators, the role of growth factors and cytokines has increasingly become more relevant in examining their effects on articular cartilage homeostasis and the development of osteoarthritis and osteoarthritis-associated pain. Catabolic factors involved in both cartilage degradation in vitro and nociceptive stimulation include IL-1, IL-6, TNF-α, PGE2, FGF-2 and PKCδ, and pharmacologic inhibitors to these mediators, as well as compounds such as RSV and LfcinB, may potentially be used as biological treatments in the future. This review explores several biochemical mediators involved in OA and pain, and provides a framework for the understanding of potential biologic therapies in the treatment of degenerative joint disease in the future.

Fibroblast Growth Factor Control of Cartilage Homeostasis

Osteoarthritis (OA) and degenerative disc disease (DDD) are similar diseases involving the breakdown of cartilage tissue, and a better understanding of the underlying biochemical processes involved in cartilage degeneration may allow for the development of novel biologic therapies aimed at slowing the disease process. Three members of the fibroblast growth factor (FGF) family, FGF‐2, FGF‐18, and FGF‐8, have been implicated as contributing factors in cartilage homeostasis. The role of FGF‐2 is controversial in both articular and intervertebral disc (IVD) cartilage as it has been associated with species‐ and age‐dependent anabolic or catabolic events. Recent evidence suggests that FGF‐2 selectively activates FGF receptor 1 (FGFR1) to exert catabolic effects in human articular chondrocytes and IVD tissue via upregulation of matrix‐degrading enzyme production, inhibition of extracellular matrix (ECM) accumulation and proteoglycan synthesis, and clustering of cells characteristic of arthritic states. FGF‐18, on the other hand, most likely exerts anabolic effects in human articular chondrocytes by activating the FGFR3 pathway, inducing ECM formation and chondrogenic cell differentiation, and inhibiting cell proliferation. These changes result in dispersed chondrocytes or disc cells surrounded by abundant matrix. The role of FGF‐8 has recently been identified as a catabolic mediator in rat and rabbit articular cartilage, but its precise biological impact on human adult articular cartilage or IVD tissue remains unknown. The available evidence reveals the promise of FGF‐2/FGFR1 antagonists, FGF‐18/FGFR3 agonists, and FGF‐8 antagonists (i.e., anti‐FGF‐8 antibody) as potential therapies to prevent cartilage degeneration and/or promote cartilage regeneration and repair in the future. J. Cell. Biochem. 114: 735–742, 2013.

Fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes

IntroductionCartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA.MethodsPrimary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity.ResultsChondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human articular chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human articular chondrocytes.ConclusionsFGFR1 is the major mediator with the degenerative potential in the presence of FGF-2 in human adult articular chondrocytes. FGFR1 activation by FGF-2 promotes catabolism and impedes anabolism. Disruption of the balance between FGFR1 and FGFR3 signaling ratio may contribute to the pathophysiology of OA.

Species-specific biological effects of FGF-2 in articular cartilage: Implication for distinct roles within the FGF receptor family

Existing literature demonstrates that fibroblast growth factor‐2 (FGF‐2) exerts opposing, contradictory biological effects on cartilage homeostasis in different species. In human articular cartilage, FGF‐2 plays a catabolic and anti‐anabolic role in cartilage homeostasis, driving homeostasis toward degeneration and osteoarthritis (OA). In murine joints, however, FGF‐2 has been identified as an anabolic mediator as ablation of the FGF‐2 gene demonstrated increased susceptibility to OA. There have been no previous studies specifically addressing species‐specific differences in FGF‐2‐mediated biological effects. In this study, we provide a mechanistic understanding by which FGF‐2 exerts contradictory biological effects in human versus murine tissues. Using human articular cartilage (ex vivo) and a medial meniscal destabilization (DMM) animal model (in vivo), species‐specific expression patterns of FGFR receptors (FGFRs) are elucidated between human and murine articular cartilage. In the murine OA model followed by intra‐articular injection of FGF‐2, we further correlate FGFR profiles to changes in behavioral pain perception, proteoglycan content in articular cartilage, and production of inflammatory (CD11b) and angiogenic (VEGF) mediators in synovium lining cells. Our results suggest that the fundamental differences in cellular responses between human and murine tissues may be secondary to distinctive expression patterns of FGFRs that eventually determine biological outcomes in the presence of FGF‐2. The complex interplay of FGFRs and the downstream signaling cascades induced by FGF‐2 in human cartilage should add caution to the use of this particular growth factor for biological therapy in the future. J. Cell. Biochem. 113: 2532–2542, 2012.

Lactoferricin mediates anti-inflammatory and anti-catabolic effects via inhibition of IL-1 and LPS activity in the intervertebral disc

The catabolic cytokine interleukin‐1 (IL‐1) and endotoxin lipopolysaccharide (LPS) are well‐known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL‐1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti‐catabolic and anti‐inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL‐1 and LPS‐mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL‐1 and LPS‐mediated proteoglycan (PG) depletion, matrix‐degrading enzyme production, and enzyme activity in long‐term (alginate beads) and short‐term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL‐1 and LPS‐mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage‐degrading enzymes, including MMP‐1, MMP‐3, MMP‐13, ADAMTS‐4, and ADAMTS‐5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor‐induced stimulation of oxidative and inflammatory factors such as iNOS, IL‐6, and toll‐like receptor‐2 (TLR‐2) and TLR‐4. Finally, the ability of LfcinB to antagonize IL‐1 and LPS‐mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. J. Cell. Physiol. 228: 1884–1896, 2013.

Biological effects of the plant-derived polyphenol resveratrol in human articular cartilage and chondrosarcoma cells.

The natural phytoestrogen resveratrol (RSV) may have therapeutic potential for arthritic conditions. RSV is chondroprotective for articular cartilage in rabbit models for arthritis, but its biological effects on human articular cartilage and chondrosarcoma cells are unknown. Effects of RSV on human articular cartilage homeostasis were studied by assessing production of matrix‐degrading enzymes (MMP‐13, ADAMTS4, and ADAMTS5), as well as proteoglycan production and synthesis. The counteractions of RSV against catabolic factors (e.g., FGF‐2 or IL‐1β) were examined by in vitro and ex vivo using monolayer, three‐dimensional alginate beads and cartilage explants cultures, respectively. RSV improves cell viability of articular chondrocytes and effectively antagonizes cartilage‐degrading protease production that was initiated by catabolic and/or anti‐anabolic cytokines in human articular chondrocytes. RSV significantly also enhances BMP7‐promoted proteoglycan synthesis as assessed by 35S‐sulfate incorporation. Protein–DNA interaction arrays suggest that RSV inhibits the activation of transcription factors involved in inflammation and cartilage catabolic signaling pathways, including direct downstream regulators of MAPK (e.g., AP‐1, PEA3) and NFκB. RSV selectively compromises survival of human chondrosarcoma cells, but not primary articular chondrocytes, revealing cell‐specific activity of RSV on non‐tumorigenic versus tumor‐derived cells. We propose that RSV exerts its chondroprotective functions, in part, by deactivating p53‐induced apoptosis in human primary chondrocytes, but not human chondrosarcoma. Our findings suggest that RSV has potential as a unique biologic treatment for both prevention and treatment of cartilage degenerative diseases. J. Cell. Physiol. 227: 3488–3497, 2012.

Lactoferricin mediates anabolic and anti-catabolic effects in the intervertebral disc

Lactoferricin (LfcinB) antagonizes biological effects mediated by angiogenic and catabolic growth factors, in addition to pro‐inflammatory cytokines and chemokines in human endothelial cells and tumor cells. However, the effect of LfcinB on intervertebral disc (IVD) cell metabolism has not yet been investigated. Using bovine nucleus pulposus (NP) cells, we analyzed the effect of LfcinB on proteoglycan (PG) accumulation, PG synthesis, and anabolic gene expression. We assessed expression of genes for matrix‐degrading enzymes such as matrix metalloproteases (MMPs) and a disintegrin‐like and metalloprotease with thrombospondin motifs (ADAMTS family), as well as their endogenous inhibitors, tissue inhibitor of metalloproteases (TIMPs). In order to understand the specific molecular mechanisms by which LfcinB exerts its biological effects, we investigated intracellular signaling pathways in NP cells. LfcinB increased PG accumulation mainly via PG synthesis in a dose‐dependent manner. Simultaneously, LfcinB dose‐dependently downregulated catabolic enzymes. LfcinBs anti‐catabolic effects were further demonstrated by a dose‐dependent increase in multiple TIMP family members. Our results demonstrate that ERK and/or p38 mitogen‐activated protein kinase pathways are the key signaling cascades that exert the biological effects of LfcinB in NP cells, regulating transcription of aggrecan, SOX‐9, TIMP‐1, TIMP‐2, TIMP‐3, and iNOS. Our results suggest that LfcinB has anabolic and potent anti‐catabolic biological effects on bovine IVD cells that may have considerable promise in the treatment of disc degeneration in the future. J. Cell. Physiol. 227: 1512–1520, 2012.

The pathophysiologic role of the protein kinase Cδ pathway in the intervertebral discs of rabbits and mice: in vitro, ex vivo, and in vivo studies.

OBJECTIVE Protein kinase Cδ (PKCδ) activation has been shown to be a principal rate-limiting step in matrix-degrading enzyme production in human articular chondrocytes. The aim of this study was to assess the role of the PKC pathways, specifically PKCδ, in intervertebral disc tissue homeostasis. METHODS Using in vitro, ex vivo, and in vivo techniques, we evaluated the pathophysiologic role of the PKCδ pathway by examining 1) proteoglycan deposition, 2) matrix-degrading enzyme production and activity, 3) downstream signaling pathways regulated by PKCδ, and 4) the effect on in vivo models of disc degeneration in genetically engineered PKCδ-knockout mice. RESULTS Studies of pathway-specific inhibitors revealed a vital role of the PKCδ/MAPK (ERK, p38, JNK) axis and NF-κB in disc homeostasis. Accordingly, in an in vivo model of disc injury, PKCδ-knockout mice were markedly resistant to disc degeneration. CONCLUSION Suppression of the PKCδ pathway may be beneficial in the prevention and/or treatment of disc degeneration. The results of this study provide evidence for a potential therapeutic role of pathway-specific inhibitors of the PKCδ cascade in the future.

Fibroblast growth factor-2 promotes catabolism via FGFR1-Ras-Raf-MEK1/2-ERK1/2 axis that coordinates with the PKCδ pathway in human articular chondrocytes.

Fibroblast growth factor 2 (FGF‐2) has been found to play an anti‐anabolic and/or a catabolic role in adult human articular cartilage via regulation of multiple signaling pathways. Upon FGF‐2 stimulation, a molecular crosstalk between the mitogen activated protein kinase (MAPK) and protein kinase C δ (PKCδ) pathways are initiated, where PKCδ positively regulates downstream MAPK signaling. In this study, we explored the relationship between fibroblast growth factor receptor 1 (FGFR1), Ras, and PKCδ in FGF‐2 signaling in human articular chondrocytes. Pathway‐specific inhibition using both chemical inhibitors and siRNA targeting FGFR1 demonstrated that, upon FGF‐2 stimulation, FGFR1 controlled both Ras and PKCδ activation, which converged on the Raf‐MEK1/2‐ERK1/2 axis. No crosstalk was observed between Ras and PKCδ. Quantitative PCR analyses revealed that both Ras and PKCδ contributed to FGF‐2‐mediated upregulation of MMP‐13, ADAMTS5, and repression of aggrecan gene. Correspondingly, FGF‐2‐mediated proteoglycan loss was effectively reversed by individual pathway‐specific inhibitor of Ras, PKCδ, and ERK1/2 in both 3‐dimensional alginate bead culture and cartilage organ culture systems. Our findings suggest that FGFR1 interacts with FGF‐2 and then activates Ras and PKCδ, which concertedly drive MAPK signaling to mediate biological effects of FGF‐2. Such an integration of dual inputs constitutes a novel mechanism of FGF‐2 signaling cascade in human articular chondrocytes. J. Cell. Biochem. 113: 2856–2865, 2012.

Bovine lactoferricin is anti-inflammatory and anti-catabolic in human articular cartilage and synovium†‡

Bovine lactoferricin (LfcinB) is a multi‐functional peptide derived from proteolytic cleavage of bovine lactoferrin. LfcinB was found to antagonize the biological effects mediated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF‐2) in endothelial cells. However, the effect of LfcinB on human articular cartilage remained unknown. Here, our findings demonstrate that LfcinB restored the proteoglycan loss promoted by catabolic factors (interleukin‐1β) IL‐1β and FGF‐2 in vitro and ex vivo. Mechanistically, LfcinB attenuated the effects of IL‐1β and FGF‐2 on the expression of cartilage‐degrading enzymes (MMP‐1, MMP‐3, and MMP‐13), destructive cytokines (IL‐1β and IL‐6), and inflammatory mediators (iNOS and TLR2). LfcinB induced protective cytokine expression (IL‐4 and IL‐10), and downregulated aggrecanase basal expression. LfcinB specifically activated ERK MAPK and Akt signaling pathways, which may account for its anti‐inflammatory activity. We also revealed that LfcinB exerted similar protective effects on human synovial fibroblasts challenged by IL‐1β, with minimal cytotoxicity. Collectively, our results suggest that LfcinB possesses potent anti‐catabolic and anti‐inflammatory bioactivities in human articular tissues, and may be utilized for the prevention and/or treatment of OA in the future. J. Cell. Physiol. 228: 447–456, 2013.