Molecular mechanisms of mechanical load-induced osteoarthritis

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that is highly expressed in OA chondrocytes compared to normal chondrocytes [3]. Its expression is directly activated by abnormal mechanical stresses. Some experiments stimulated articular cartilage through mechanical overloading in vitro [4] (cyclic compression stimulation (CTS), 6% compression (36 kPa) at 1HZ for 4 h) and in vivo [5] (rats on treadmill five days a week for 60 minutes daily (at a speed of 20 m/min)) and observed a significant upregulation of IL-1β expression. IL-1β mediates OA progression by activating downstream signaling pathways. In addition, IL-1β inducible nitric oxide synthase (iNOS) expression increases nitric oxide (NO) concentration and causes oxidative stress [6]. This cytokine also induces the expression of cyclooxygenase-2 (cox-2) and upregulates prostaglandin E2 (PGE2) concentrations. This enzyme mediates bone synthesis that may be associated with osteophyte formation during OA progression [7]. Furthermore, IL-1β activates all three MAPK pathways (ERK, p38, and JNK). Activation of these pathways generates several matrix-degrading enzymes such as MMP1, MMP3, MMP13, ADAMT4, ADAMT5, and aggrecanase that activate downstream inflammatory factors such as COX-2, iNOS, PGE-2, and IL-6 [8]. Through the nuclear factor kappa-B (NF-κB) pathway activation, downstream matrix degrading enzymes such as MMP1 and MMP13 and inflammatory factors including tumour necrosis factor-α (TNF-α), NO, COX-2, and IL-6 are also upregulated by this cytokine [9]. In addition, IL-1β triggers the Wnt pathway and induces MMP expression [10]. IL-1β upregulates MMP3 and MMP9 by activating the JNK pathway to accelerate synovial senescence [11]. It also induces iNOS and COX-2 by activating PI3K and Akt pathways to upregulate the concentration of NO, PGE2, MMPs, and ADAMTs [12]. IL-1β enhances nuclear factor erythroid-2 related factor 2 (Nrf2) and Heme Oxygenase-1 (HO-1) to upregulate matrix-degrading enzymes and oxidative stress [13]. IL-1β can upregulate MMP3 and MMP9 and ADAMTs by activating IL-17, thereby promoting joint inflammation and inducing chondrocyte apoptosis [14]. Ohtsuki et al. [15] observed that IL-1β is also involved in upregulating Cell migration-inducing hyaluronidase 1 (CEMIP), which induces the degradation of hyaluronan in ECM. Several miRNAs (miR-34a, miR-30a, miR-145, and miR-27a) are activated by IL-1β and participate in progression of OA by activating matrix-degrading enzymes and downstream inflammatory pathways such as MAPK and NF-κB [16]. Notably, IL-1β directly induces the activation of matrix-degrading enzymes, resulting in cartilage damage. Wang et al. [17] showed a significant decrease in the friction coefficient of cartilage after inhibiting IL-1β in OA model rats (IL-1Ra, 500 μg/ml in a total volume of 40 μl per intra-articular injection) and vitro (20 ng/ml, 36 h). These frictions promoted OA progression and upregulated IL-1β concentration. Clinical applications of IL-1β inhibitors against OA can achieve significant effects. IL-1β also stimulates OA pain. The Pannexin-1 (Panx1) channel is an important target for inducing mechanical pain in OA. Activating the Panx1 channel in mouse microglia releases IL-1β, thereby causing mechanical pain. In addition, upregulated IL-1β participates in inducing OA pain by activating neurotrophin (NGF) (Fig. 2) [18].

The tumour necrosis factor-α (TNF-α) pathway has been reported to be actively involved in the pathogenesis of osteoarthritis that is induced by mechanical loading. This pathway is activated through excessive mechanical load in vivo (90 g compressive mechanical loading was applied to rats for 96 h) and in vitro (CTS;36 kPa at 1HZ for 4 h) that triggers downstream inflammatory pathways to induce chondrocyte apoptosis and ECM degradation [19]. Studies have shown that TNF-α directly induces apoptosis and/or necrosis of rat chondrocytes in vitro (recombinant human 5 ng/ml) and activates matrix-degrading enzymes such as MMP1, MMP13, and ADAMT4/5 [20]. Li et al. [21] reported that activated TNF-α pathway upregulated IL-1β. TNF-α also activates the NF-κB pathway, MAPKs, and c-jun pathway and indirectly upregulates matrix-degrading enzymes such as collagen type 10 (Col10), MMP13, ADAMT5, and ADAMT9 that caused articular cartilage degradation [22]. Furthermore, NOD (nucleotide binding oligomerization domain)-like receptors (NLRP3) play an important role in the TNF-α pathway activation induced by mechanical load [23]. In their study, Grodzinsky et al. [24] documented that after inhibiting TNF-α pathway in chondrocytes, the level of inflammatory factors in articular cartilage was significantly downregulated, thereby alleviating ECM degradation and chondrocyte apoptosis (Fig. 3).

In vivo (rats on treadmill, 20 m/min for 45 minutes daily for four weeks) and in vitro (10% CTS, 0.5 Hz, 12 h) experiments have shown that overloading significantly upregulates the NF-κβ pathway and its downstream inflammatory factors [25], such as TNF-α, IL-1, IL-4, IL-6, and NO. It also activates matrix-degrading enzymes such as MMP3, MMP9, and MMP13 and ADAMT4, ADAMT5, and ADAMT9, thereby, enhancing OA progression [26]. Yang et al. [27] reported that physiological mechanical stress downregulated the NF-κβ pathway and reduced the inflammatory responses of chondrocytes. Inhibiting the NF-κβ pathway can effectively reduce overload-induced osteoarthritis incidences [28]. Chang et al. [29] reported that Gremlin-1, an extracellular antagonist of bone morphogenetic proteins (BMPs), is a key regulator of the NF-κβ pathway in overload-induced OA. A study by Suzuki et al. [30] found that overload-induced NF-κβ pathway significantly upregulated cathepsin K in cartilage matrix degradation.

In addition, NF-κβ activates downstream oxidative stress signals that indirectly induce ECM degradation and chondrocyte apoptosis. The NF-κβ pathway activates iNOS to upregulate NO levels in chondrocytes [31]. Excessive reactive oxide species (ROS) causes chondrocyte DNA mismatches, protein oxidative modification, and cell membrane lipid peroxidation that eventually result in chondrocyte apoptosis. The NF-κβ pathway also induces COX-2 and downregulates PGE2 to promote inflammation. NF-κβ and IL-1β pathways have a synergistic effect in the progression of OA. High IL-1β expression activates the NF-κβ pathway to induce chondrocyte apoptosis. Furthermore, NF-κβ upregulates downstream inflammatory cytokines such as hypoxia inducible factor-2α (HIF-2α), E74-like factor 3 (ELF3), SOX9, BMP2, IL-6, and TNF-α that promote chondrocyte apoptosis and ECM degradation [32]. Murahashi et al. [33] observed that overload-induced NF-κβ activates HIF-2α signals and upregulates downstream MMP13 and ADAMT5. This promotes cartilage matrix degradation and HIF-2α pathway. Furthermore, the Fas pathway is activated by NF-κβ to induce chondrocyte apoptosis. The overexpression of SOX9 downregulates OA factors such as MMPs and IL-1, which have been proven to induce OA in vivo and in vitro. The expression of SOX9 is antagonized by NF-κβ pathway in OA cartilage [34]. Besides, NF-κβ activates the ELF3 pathway and downstream MMPs causing cartilage matrix degradation [32]. The ELF3 pathway inhibits the SOX9 activator, thereby inhibiting the synthesis of SOX9-driven COL2A1, and promotes the progression of post-traumatic osteoarthritis (Fig. 4) [35].

Wnt signals include canonical β-catenin-dependent and non-canonical β-catenin-independent signaling pathways. Wnt signals of different types and strengths regulate each other and maintain a delicate balance. Wnt16 signaling inhibits Wnt3a from over-activating the typical Wnt pathway, thereby preventing OA risk factors such as chondrocyte apoptosis and lubricin reduction [36].

It has been shown that under the same mechanical load, Wnt5a knockout rats are more susceptible to OA [37]. A study by Yuan reported that Wnt pathway inhibition by overload is characterized by OA manifestations such as induced ECM degradation, upregulated inflammatory factors, and trabecular bone erosion. The activation of the Wnt pathway can reverse these symptoms [38]. Physiological stress can activate Wnt signaling to inhibit osteoclast production and promote COL2A1 synthesis [39]. Wnt3a-RANKL signaling plays an antagonistic role in ECM degradation while Wnt16 plays a protective role by activating the PCP-mTORC1-PTHrP axis (a signal regulating cell proliferation) that inhibits ECM degradation [40]. The Wnt/β-catenin pathway significantly downregulates downstream coding products (MMP13 and ADAMT5) by regulating specific OA-related microRNAs such as miR-27a/b, miR-140, miR-146a/b, and miR365 [41]. The physiological mechanical load induces the upregulation of the Wnt5b/9a pathway thereby activating proteoglycan 4 (Prg4) to induce lubricin expression in the superficial area of articular cartilage [37], which significantly reduces joint friction.

Moreover, abnormal activation of the Wnt signal plays a role in cartilage destruction. Wnt1/3a signals that are induced by overloading significantly upregulate matrix degradation enzymes such as MMP13 and ADAMT5, ADAMT7, and ADAMT7 [40]. Wnt-5a phosphorylates JNK and PKC by activating atypical Wnt pathways thus inducing chondrocyte apoptosis. The overexpression of Wnt5a significantly upregulates MMPs and ADAMTs, thereby inducing chondrocyte apoptosis [42]. A study by Huang et al. reported that MIR-29a, a Wnt1 pathway transcription factor, upregulated caspase-3 expression causing chondrocyte apoptosis [43]. Moreover, Deshmukh et al. [44] observed that injecting SM04690 (a strong Wnt signal blocker) into a joint with a high expression of the Wnt signal effectively alleviated the overloading-induced damage [45]. Excessive Wnt signal levels upregulates ADAMT4, ADAMT5, ADAMT7, and ADAMT12 and promotes the degradation of ECM. However, IWP-2 (a Wnt signaling inhibitor) suppresses overloading-induced chondrocyte catabolism [46].

Different types and intensities of Wnt signals exert distinct effects on the articular cartilage. This may explain the different effects of excessive load and moderate load on the survival of articular cartilage. Moderate Wnt signal induced by physiological stress maintains the normal survival of articular cartilage. Meanwhile, excessive mechanical loads result in the abnormal expression of Wnt signal and induces changes associated with osteoarthritis such as chondrocyte apoptosis, matrix destruction, and osteophyte formation (Fig. 5).

Upregulation of transforming growth factor-β (TGF-β) in chondrocytes antagonizes chondrocyte apoptosis and matrix degradation caused by excessive mechanical load in vitro (36 kPa CTS at 0.5HZ for 2 h) and in vivo (OA model rats) [47]. TGF-β maintains functions of articular cartilage by transducing Smad2/3P signals thereby promote collagen (Col2a1) and fibronectin (Fn1) synthesis and prevent the degradation of cartilage matrix induced by overload [48]. Moreover, TGF-β inhibits MMP13 activation by stimulating IL-2/12 [49]. It has also been reported that TGF-β inhibits cartilage matrix degradation by upregulating SOX9 signals. 2,3′-Phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2; one of TGF-β transcripts) regulates the physiological synthesis of proteoglycans in ECM [50]. Boyan et al. [51] showed that activating the TGF-β pathway with 25-hydroxyvitaminD3 [25 (OH) D3] can inhibit the IL-1β inflammatory pathway, antagonize chondrocyte apoptosis, and downregulate matrix-degrading enzymes. Elsewhere, Chavez et al. found that Prg4, one of the transcripts of TGF-β, promoted the secretion of articular cartilage lubricant to reduce the damage of articular cartilage caused by mechanical friction [52]. In addition, TGF-β confers anti-OA effect by activating some MIRNAs. Of note, TGF-β downregulates MIR-92a to inhibit adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) and p38 pathway whereas it upregulates MIR-135b and MIR-140-5p to prevent chondrocyte apoptosis and cartilage matrix degradation (Fig. 6) [53].

Numerous studies have reported that mechanical loading causes differential expression of microRNA (MIRNA) in articular cartilage and regulates the levels of matrix-degrading enzymes and inflammatory factors in osteoarthritis cartilage [54]. Specifically, it targets the expression of downstream transcripts. A study by Yang et al. [55] found that mechanical loading upregulated MIR-365 expression in chondrocytes. In addition, another study showed that MIR-365 overexpression activated the IL-1β pathway and increased the expression of matrix degrading enzyme (MMP13) and collagen type X (ColX). A study by De Palma et al. [56] reported that mechanical loading downregulated MIR-155 and MIR-181a in OA cartilage and activated the NF-κB pathway. Mechanical loading increased MIR-590-5p expression and activated TGF-β thereby enhancing chondrocyte apoptosis and autophagy [57]. Overloading activates the IL-1 β pathway and ADAMT5 and upregulates MIR-145 to promote ECM degradation [16]. Mechanical load upregulates MIR-365 in chondrocytes and promotes the expression of osteogenic genes and induce osteoarthritis osteophyte formation. Overloading upregulates MIR-204 expression, which then activates NF-κB and blocks the synthesis of proteoglycans, thus affecting ECM anabolism [58]. Overloading also upregulates miR-125a-5P, which in turn activates Smad2 to protect against Osteoarthritis [59]. Cheleschi et al. [41] indicated that mechanical loading upregulated MIR27a/b, MIR-140, and MIR-146a/b and downregulated MIR-365 expression, resulting in high expression of MMP13, ADAMT5, and HDAC-4. Changes in MIRNAs induced by mechanical loading do not always cause damage to chondrocytes. A study by Guan et al. [60] found that physiological mechanical load inhibited the Notch1, IL-6, and IL-1 signaling pathways and upregulated MIR-146a to alleviate osteoarthritis. It also upregulates MIR-204 and MIR211 leading to decreased expression of matrix-degrading enzymes in chondrocytes. It activates downstream Runx2 signals that maintain ECM homeostasis [61].

Several recent studies have revealed that oxidative stress plays an important role in mechanical stress-induced OA. The ROS in chondrocytes is mainly produced by the mitochondrial electron transport chain. A study by Coleman et al. [62] reported that overload (1.0 MPa, 0.5 Hz) on bovine articular cartilage decreased proton membrane potential, respiratory activity and ATP production of chondrocytes, and elevated ROS production. It was reported that levels of antioxidant enzymes such as SOD, CAT, GSH, LDHA, and GPX in the cartilage of OA patients were significantly lower than those in normal articular cartilage, and excessive ROS level altered the redox balance in chondrocytes [63]. Excessive ROS generation can damage DNA and protein and cause lipid peroxidation in chondrocytes. ROS induces mitochondrial dysfunction and activates pro-inflammatory mediators and matrix degradation enzymes, resulting in OA. ROS has been found to participate in several inflammatory pathways in chondrocytes. Excessive ROS inhibits the PI3K/Akt pathway and activates MEK/ERK-MAPK/JNK pathway to upregulate caspase-3 and thereby causing chondrocyte apoptosis. Excessive ROS induced by overload activates inflammatory factors such as NF-κβ, protein kinase C, TNF-α, and IL-1β and eventually activates downstream iNOS, IL-8, and COX-2. Besides, excessive ROS activates matrix degradation enzymes such as MMP3, MMP9, MMP13, ADAMT4, and ADAMT5 in chondrocytes and degrade ECM components such as collagen type II, aggrecan, and proteoglycans [64]. Besides, antioxidants reduce overload-induced osteoarthritis which hence can be used for the clinical treatment of OA. Application of N-acetylcysteine (a common antioxidant) to overloaded chondrocytes effectively reversed the expression of MMPs and ADAMT4 and downregulation of type II collagen in chondrocytes of OA rats induced by mechanical load [65].

These mechanical stress-induced signal pathways are not isolated from each other. For example, IL-1β, TNF-α, NF-κB, and ROS activate each other and promote cartilage inflammation. Some experiments confirm that IL-1β, TNF-α, NF-κB, and ROS can downregulate TGF-β in articular cartilage. Different Wnt signal factors play different roles in mediating OA. For example, IL-1β, TNF-α, NF-κB, and ROS all upregulate Wnt5a/9a level, while inhibiting the expression of Wnt3a/16. Overexpression of Wnt3a/16 suppresses the levels of IL-1β, TNF-α, NF-κB, and ROS. Different types of microRNAs have completely different effects on the OA-related inflammatory factors (Table 1 and Fig. 7).

There are receptors in cartilage that detect mechanical stress and regulate downstream signal pathways. Integrin (a cell adhesion molecule), for example, detects the mechanical signal in ECM and then activates the integrin-FAK-MAPKs axis to the upregulate OA-promoting factors such as MMPs, ADAMTs, and IL-6. Besides, integrin-fibrin-α5β1 axis detects mechanical signals and increases the expression of matrix degrading enzymes, MMP3, and MMP13 [66]. Hirose et al. [67] showed that integrin inhibitor cilengitide significantly downregulated several inflammatory factors such as IL-1β, TNF-α, MMPs, p-FAK, p-ERK, JNK, and p-p38 that were upregulated by overloading.

Moreover, ion channels play important roles in mechanical signal transduction. Mechanical signals are converted into electrical signals which then activate inflammatory signal pathways. A study by Lee et al. [68] observed that mechanical load generated Ca2+ transient using an atomic force microscope. Blocking this calcium transient with GsMTx4 (a piezoresistive peptide) significantly inhibited the apoptosis of chondrocytes after mechanical injury. Elsewhere, Xu et al. [69] reported that the mechanosensitive ion channel transient receptor potential channel 4 (TRPV4) of chondrocytes could detect mechanical stress and induce extracellular Ca2+ influx thereby upregulating levels of fas-related proteins and caspase-3, caspase-6, caspase-7, and caspase-8 triggering chondrocyte apoptosis. Some studies reported that voltage-dependent calcium channels are involved in the perception of persistent OA pain by modulating the CGRP and AC-PKA/PKC/MAPK signaling pathways in the dorsal root ganglion [70]. Besides, TRPA1 and RPV4 are closely associated with modulation of mechanical pain in OA. Mechanical loading activates matrix-degrading enzymes such as ADAMT5, ADAMT9, and MMP13 through TRPV1 and TRPV4 ion channels [71]. It has been reported that P2X7 activates Ca2+ and K + channels in chondrocytes under mechanical loading, causing chondrocyte injury [72].

Primary cilia are a key mechanical signal transduction factor in chondrocytes, which is a cytoskeletal organelle that extends from the cell surface into the external environment of the cell. Primary cilia are sensitive to mechanical stress on chondrocytes; when exposed to cyclic tensile strain, its length significantly shortens to absorb the stimulation of mechanical stress on chondrocytes. The main biological activity of primary cilia is the intraflagellar transport (IFT), a process in which primary cilia form microtubules so that soluble proteins and transmembrane receptors move along the microtubules [73]. In addition, primary cilia are also involved in mechanical load-related growth plate chondrocyte development. The dysfunction of primary cilia significantly impairs mechanical signal transduction. Some experiments have shown that animals with primary cilia damage, such as IFT-80 and IFT-88 knockout rats, have lower mechanical signal transduction efficiency and develop OA earlier [74].

Some other receptors in chondrocytes sense mechanical load. Gremlin-1 (an extracellular antagonist that secretes protein and bone morphogenetic protein (BMPs)) has been proposed to play a key role in perceiving mechanical load. Intra-articular injection of gremlin-1 antibody or chondrocyte-specific deletion gremlin-1 in OA model mice significantly reduces the sensitivity of articular cartilage to mechanical load [29]. Furthermore, hydrogen peroxide-inducible clone-5 (Hic-5) is considered to be a mechanically sensitive adaptor. The catabolic gene expression of chondrocytes induced by overloading significantly decreases after Hic-5 knockout [75].

Pain is the most prominent clinical symptom of osteoarthritis, which is also the main reason for patients to seek medical treatment. Long-term abnormal mechanical load induces structural changes in weight-bearing joints, which lead to joint pain. MRI studies have been used to confirm correlations between OA pain and these changes, including cartilage loss, osteophyte formation, and synovitis [76]. In addition, OA pain is also closely related to joint inflammation exacerbated by abnormal mechanical load, anti-inflammatory treatments, including glucocorticoids, and IL-1β/TNF-α inhibitors, which can effectively relieve pain symptoms during walking. In addition, there are multiple nociceptors in OA joints, which receive stimulus including mechanical/chemical/inflammatory mediators and convert harmful signals into pain. The transient receptor potential (TRP) family, possessing transmembrane domains, plays an important role in signal reception and conversion, which functions as ion channels when stimulated [77]. Some recent evidence suggests that nerve growth factor (NGF) increases the sensitivity of nociceptors in joints to mediate OA pain. NGF inhibitors can effectively alleviate OA pain [78]. A report by Carcolé et al. [79] indicated that sigma-1 receptors perceived and modulated mechanical pain in osteoarthritis. Furthermore, OA patients usually have peripheral nociceptors, a phenomenon in which the sensitivity of receptors increases and the excitability threshold of pain nerves decreases, which is clinically manifested as hyperalgesia (physiological stimulation can also cause pain).

There are already quite a few promising molecular targeted therapies for OA clinically. It is reported that IL-1β inhibitors, such as Diacerein/AMG108/anakinra, effectively protected chondrocytes through reducing MMPs and ROS and preventing ECM degradation both in vitro (Diacerein, 20 μg/ml) and in vivo (rats, Diacerein at 5, 15 or 50 mg/kg) [34]. Adalimumab, a TNF-α inhibitor, has been shown to be efficacious in relieving the pathological symptoms of RA in clinic (DE001/003). However, it was less efficacious in reducing symptoms and inflammation factors of OA in a RCT(NCT00597623) compared with RA [80]. An iNOS inhibitor (SD-6010) had achieved significant effects on relieving OA symptoms of an RCT (NCT00565812). P38-MAPK pathway inhibitors (PH-797804) have also been verified effective anti-inflammatory and symptom relief in vitro (50 ng/ml) and vivo (rats,1 μM/kg) [81]. In addition to these, many other cytokines associated with OA have also become therapeutic targets. Some siRNA targeting pro-inflammatory mediators (including TNFα, IL-1β, IL-6, IL-18, and NF-κβ) effectively reduced the inflammation of OA [82].