Molecular Mechanisms of Osteoarthritis

Osteoarthritis (OA) is a chronic degenerative disorder which occurs when the cartilage that cushions the ends of bones in joints wear down over a period of time. The molecular mechanisms of osteoarthritis have not received much attention among the public, except the few who are in research and development of drugs. OA is the most prevalent chronic joint disease, which increases in prevalence with age and affects majority of persons over 65.
Unlike Rheumatoid arthritis (RA), OA is caused by mechanical wear and tear at the joints. Also, while RA can occur at any stage, OA occurs later in life of the individual. Both however, share some similarities in their symptoms. But, while RA affects both sides of the affected joints, OA can affect one side of the joint.

Symptoms of Osteoarthritis

Symptoms of Osteoarthritis may develop slowly and worsen over a period of time. Some of the signs and symptoms include:
• Loss of flexibility at the affected joints.
• Pain at the affected joint.
• Stiffness of joints especially upon awaking.
• Swelling.
• Tenderness of tissues around the affected joints.

Pathological Progression of Osteoarthritis

The pathological process of osteoarthritis begins by an alteration in the composition and organization of the extracellular matrix. When this happens, the articular chondrocytes exhibit a transient proliferative response and increased matrix synthesis (Col2, aggrecan etc) in an attempt to initiate repair of damage caused by pathological stimulation.
Changes in the composition and structure of the cartilage further stimulate chondrocytes to produce more catabolic factors involved in cartilage degradation. As a result, the proteoglycans and collagen network break down, leading to the disruption of the cartilage integrity. Later on, the chondrocytes will undergo apoptosis and the articular cartilage will be completely lost. The molecular mechanisms of osteoarthritis involve several pathways that network with each other to induce inflammatory response in osteoarthritis patients and include growth factors and cytokines.


Growth Factors and Osteoarthritis

Transforming growth factor B (TGF-B) plays important role in skeletogenesis and inhibits chondrocyte hypertrophy and maturation. The inhibition of TGF-B represents a potential mechanism in the development of OA, and the loss of TGF-B is associated with cartilage damage. Some other growth factors that play important roles in cartilage homeostasis are fibroblast growth factor-2 and 18 (FGF-2 and FGF-18). FGF-2 has a potent catabolic and anti-anabolic role in human cartilage homeostasis. It is released in supraphysiological amounts during loading/ injury of the cartilage matrix. It activates multiple transduction signal pathways (MAPKs) such as ERK, p38 and JNK. FGF-2 can stimulate MMP-13 expression, which is the major degrading enzyme to type II collagen. Its stimulation also mediates the up-regulation of matrix-dependent enzyme expression (ADAMTS-5 and MMP-13) and the down-regulation aggrecan expression. But PKCგ inhibition significantly impairs the detrimental effects mediated by FGF-2.
FGF-18 which is a secreted heparin-binding polypeptide growth factor is involved in cartilage growth and maturation and is implicated in the development of functional cartilage and bone tissue. It has roles in enhancing regeneration and repression of damaged cartilage by stimulating chondrogenesis.

Molecular Mechanism of Wnt/β-catenin Signaling in Osteoarthritis

Wnts are a family of extracellularly secreted glycoproteins that are in numerous biological activities, including cell proliferation, differentiation, polarization and fate determination. They have been implicated in the development of some diseases through canonical β-catenin-dependent and noncanonical β catenin-dependent signaling pathways.
During cartilage development, Wnt/β-catenin signaling activity is strictly regulated in chondrogenesis and chondrocyte maturation. Excessive Wnt pathway activation in the adult articular cartilage under IL-1β stimulation is thought to be an OA progression susceptibility factor. An increase in expression of Wnt1 pathway activator, Wnt inducible-signaling pathway protein 1 (WISP-1) induces articular cartilage degradation through up-regulation of the expression MMPs and aggrecanases in chondrocytes and macrophages. But the expression levels of some wnt pathway antagonists, such as sclerostin, dickkopf WNT signaling pathway inhibitor 1 (DKK1), and secreted frizzled-related protein 3 (sFRP3) usually decreases in parallel with OA progression. Therefore, an up-regulation of the expression of these antagonists alleviates OA cartilage destruction. A mutant in sFRP3 causes increased levels of active β-catenin, thereby promoting abnormal articular chondrocyte hypertrophy and results in hip and knee in OA patients.

Molecular Mechanism of the PI3K/AkT/mTOR Pathway in OA Pathogenesis

The PI3/AkT/mTOR pathway plays significant roles in the molecular mechanisms of osteoarthritis in three aspects of OA which are cartilage, synovial inflammation and subchondral bone sclerosis.

Cartilage Homeostasis

Cartilage homeostasis is important to articular health and is defined as the state in which synthesis of extracellular matrix (ECM) is balanced by its degradation. The destruction of cartilage homeostasis initiates and boosts OA pathogenesis. It is marked by elevated MMPs, a disintegrin and metalloproteinases with thrombospondrin motifs (ADAMTSs) and a reduced collagen II and aggrecan levels.
The PI3K/AkT pathway plays a role in both ECM anabolism and catabolism. When stimulated, AkT promotes the synthesis of collagen II and an over-expression of AkT enhances proteoglycan synthesis in human chondrocytes under the stimulation of tert-butylhydroperoxide (tBHP). When PI3k is activated, Akt is phosphorylated, which promotes the activation of mTORC1. The activated mTORC1 inhibits autophagy by associating with ULK1- Atg13-FIR200 complex, thereby inhibiting autophagosome formation. Inhibiting of mTOR and the subsequent increase in autophagic activity may restore homeostasis in articular cartilage chondrocytes.

NF-kB in OA Pathogenesis

When excessively activated, NF-kB signaling up-regulates hypoxia-inducible factor 2α (HIF-2α) transcription. This results in enhanced OA progression through further induction of catabolic factors such as MMPs, VEGF and IHH. But a moderate activity of NF-kB is required to maintain healthy articular cartilage.
The NF-kB-HIF-2α axis may be a potential therapeutic target. This is because a moderate dose of an IKK inhibitor can suppress HIF-2α expression without significant effect on cell survival.


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Nuclear Factor-Kappa B (NF-kB)

Nuclear factor-kappa B (NF-kB) is a transcription factor that plays critical functional roles in inflammation, immunity, cell proliferation, differentiation, and survival. It exists in an inactive state in the cytosol and can be stimulated by molecules such as TNFα, and other cell stressors.

NF-kB is found in almost all cellular cell types and is identified as a regulator of kB light chain expression in mature B and plasma cells.

Because NF-kB has the ability to influence the expression of numerous genes, its activity is tightly regulated at multiple levels. The primary mechanism for regulating NF-kB is through inhibitory IkB proteins and the IKK complex, which phosphorylates IkBs.

Nuclear Factor-kappa B (NF-kB) Activation

NF-kB activation is initiated by TNFα. When TNFα binds to TNF receptors and activate them, IkB kinase (IKK) is ultimately triggered, which leads to the phosphorylation of IkB. The phosphorylation of IkB results in ubiquitination and degradation of IkB. When this happens, the remaining NF-kB dimmer (e.g., p65/p50 or p50/p50 subunits) translocates to the nucleus, where it then binds to a DNA consensus sequence of target genes.


Nuclear Factor-kappa B (NF-kB) Structure-Function relationship

NF-kB is a multiple-gene family of proteins that can form stable homo- and heterodimeric complexes, which vary in their DNA binding specificity and transcriptional activation potential.

There are five proteins of the NF-kB family in mammalian cells; they are RelA (P65), C-Rel, RelB, and NF-kB1 (p50 and its precursor p105), and NF-kB2 (p52 and its precursor p100). NF-kB and Rel proteins share a highly conserved 300 amino acid N-terminal Rel homology domain (RHD), which is responsible for DNA binding, dimerization, and association with IkB inhibitory proteins.

The p50/p65 complex shows strong transcriptional activation, whereas p50/p50 and p52/p52 homodimers suppress transcription of NF-kB target genes.


Nuclear Factor-kappa B (NF-kB) Is Inhibited By IkB Family Proteins

NF-kB is inhibited by IkB family proteins through NF-kB/IkB complex formation. There are seven inhibitory protein members of IkB family. They are IkBα, IkBβ, IkBε, IkBϒ, Bcl3, NF-kB1 precursor and NFkB2 precursor.

The IkB family members have a common ankyrin repeat domain. They regulate the subcellular localization, and hence, the DNA binding and transcriptional activity of NF-kB proteins.


IkB Regulates NF-kB Translocation to the Nucleus.

NF-kB is localized in the cytoplasm in an NF-kB/IkB complex, which is inactive. The inactive NF-kB/IkB co2mplex is a result of masking of the nuclear localization signals (NLS) on the NF-kB subunits by the IkB proteins. Hence, the degradation of IkB would lead to unmasking of the NLS, allowing NF-kB to undergo translocation to the nucleus.

The IkB proteins show a preference for specific NF-kB/Rel complexes, which provides a means to regulate the activation of distinct Rel/NF-kB complexes.

After the binding and transcriptional activity of NF-kB on DNA, it induces the expression of IkBα, which enters the nucleus and remove NF-kB  from DNA by forming NF-kB/IkB complex with the released NF-kB. The complex is then expelled from the nucleus back to the cytoplasm as a result of potent nuclear export signals on IkB and p65.


IkB Degradation Is Mediated By Ubiquiti/Proteasome System

The activation of NF-kB is achieved through the signal induced proteolytic degradation of IkB. This degradation is initiated by the stimuli dependent phosphorylation of IkB at specific N-terminal residues (S32/S36 for IkBα, S19/S23 for IkBβ), and is mediated by the ubiquitin/proteasome system.

Phosphorylation of IkB however, is not enough to initiate degradation of IkB. Ubiquitination and subsequent degradation depends on the recognition of phosphorylated IkB by the β-TrCp, an F-box/WD containing component of the Skp1-cullin-F-box (SCF) class of E3 ubiquitin ligases.


IKKs Mediates The Phosphorylation Of IkB On Serine 32 and 36

Phosphorylation of IkBa on the Serine 32 and 36 is mediated by IkB kinases (IKKs), whose activity is induced by activators of the NF-kB pathway. IKK contains two subunits, IKK1 (IKKα) and IKK2 (IKKβ). It also contains a regulatory subunit, NEMO ( IKKy, IKKAPI, FIP3).

IKK1 and IKK2 are homologous kinases, and both contain an N-terminal kinase domain and a C-terminal region with two protein interaction motifs, a leucine zipper (LZ), and ahelix-loop-helix (HLH) motif.

The LZ domain is responsible for demerization of IKK1 and IKK2. It is also essential for IKK complex activity. The IKK1/2 complex associates with NEMO through a short interaction motif located at the C-terminus of either catalytic subunit. Short peptides derived from the interaction motif can be used to disrupt the IKK complex and prevent its activation. NEMO connects the IKK complex to upstream activators through its C-terminus, which contains a zinc finger motif. NEMO also undergoes stimulus dependent interaction with components of TNF receptor complex.





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The Functions of Zinc in Immune System

Zinc is an essential macronutrient, which play a crucial role in multiple cellular functions, including immune cell signaling. In zinc dyshomeostasis, which includes zinc deficiency, there are impairments in several cellular and organ function including overall immune function and increased susceptibility to infection. This shows that zinc cannot be overlooked in immune system and other cellular processes.

Some of the problems associated with zinc deficiency are growth retardation, neurological disorder, immune dysfunction and Acrodermatitis enteropathica, a metabolic disorder.


Zinc Transporters

Zinc coordinates its signaling through two families of zinc transporters and metallothiones. The two families of zinc transporters are:

The Solute-linked carrier 39 (SLC39A or ZIP) family of zinc transporters, which transport zinc into the cytosol and out of the intracellular organelles and,

The Solute-liked carrier 30 (SLC30A or ZnT) family of zinc transporters, which transport zinc out of the cytosol and into the intracellular organelles.

Both ZIP and ZnT transporters are expressed in a cell- or tissue-specific manner.

Metallothione (MT)

Metallothione is a zinc-binding protein that functions as a reservoir of intracellular zinc. It has the ability to bind up to seven zinc ions per MT molecule. It also plays a crucial role in the distribution, transport and maintenance of intracellular zinc ions.

The Function of Zinc in Pathogen Invasion

Zinc regulates complex signaling pathways in immune cells. As a result, when an invasion by a pathogen occurs, the pathogen creates a conflict in which Zn becomes a shared resource.

In this battle-like state, the pathogen strives to utilize Zn for its biological functions at the expense o the host, while the host cells seek to reserve Zn and render it inaccessible for pathogen uptake. This strategy of the host cell curtails the growth of some pathogens, but there are some pathogen which resist this cellular mechanism by possessing strong Zn acquisition machineries that effectively compete with the host for Zn.

Excess zinc however, can exert toxic effects on microbial survival. And the immune cells have taken advantage of this to localize and fuel excessive Zn concentrations that intoxicate the pathogen without impacting host cells.


Zinc Signals in Monocytes and Macrophages.

The immune system provides two layers of defense against pathogens; they are innate and adaptive immunity. Innate immunity, which is the frontier of host defense, involves the recognition of pathogen-associated molecular patterns (PAMPs), conserved structures of invading pathogens, and the immediate initiation of immune responses.

During invasion, mononuclear phagocytes of innate immunity immediately recognize invading pathogens through the sensing of PAMPs by pathogen-recognition receptors (PRR), including Toll-like receptors (TLRs). Upon PAMP engagement, individual TLRs differentially recruit adaptor molecules such as MyD88, TRIF, TIRAP-dependent NF-kB, MARK, PI3K, and the TRIF/TRAM-dependent IRF3 pathway, and elicits a variety of monocyte and macrophage effectors’ functions.


TRIF/TRAM-dependent pathway

Signaling through TRIF activates several transcription factors, including NF-kB, IRF3, and AP-1. This leads to the production of cytokines and type-1 IFN, as well as maturation of myeloid dendric cells. Biological responses from TRIF-dependent signaling depends on both the type of cell responding and the particular TLR that is activated.

In TLR4 signaling, the TLR4 TIR domain use TRAM to recruit TRIF to the signaling complex, either by operating from the plasma membrane or from the endosomes. Localization of TRAM to the endosomes is necessary for IRF3 activation in the TRIF-dependent pathway.

When a lipopolysaccharide binds to TLR4, it leads to rapid zinc influx into the cytoplasm of monocytes and macrophages, which triggers zinc-mediated regulation of major signaling pathways, including TRIF/TRAM pathway.


MyD88/TIRAP-dependent NF-kB pathway.

The NF-kB transcription factor is a central regulator of proinflammatory gene induction and functions in a variety of immune responses. It influences the expression of proinflammatory cytokines, Chemokines, acute phase proteins, matrix metalloproteinase, adhesion molecules, growth factors, and other factors involved in inflammatory responses.

Zinc regulates the NF-kB activity by suppressing LPS-induced activation of IKKB. This is through a mechanism that is initiated by the inhibition of cyclic nucleotide phosphodiesterase (PDE), and subsequent elevation of cGMP, cross-activation of protein kinase A (PKA), and inhibitory phosphorylation of protein kinase Raf-1.

Another mechanism which involves direct inhibition of IKK upstream of NF-kB is mediated by ZIP8, which increase intracellular zinc, and involves a direct binding of Zn to IKKB.