Objectives Examine whether osteoarthritis (OA) progression can be delayed by halofuginone in anterior cruciate ligament transection (ACLT) rodent models. Methods 3-month-old male C57BL/6J (wild type; WT) mice and Lewis rats were randomised to sham-operated, ACLT-operated, treated with vehicle, or ACLT-operated, treated with halofuginone. Articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)-modified Mankin criteria. Immunostaining, flow cytometry, RT-PCR and western blot analyses were conducted to detect relative protein and RNA expression. Bone micro CT (μCT) and CT-based microangiography were quantitated to detect alterations of microarchitecture and vasculature in tibial subchondral bone. Results Halofuginone attenuated articular cartilage degeneration and subchondral bone deterioration, resulting in substantially lower OARSI scores. Specifically, we found that proteoglycan loss and calcification of articular cartilage were significantly decreased in halofuginone-treated ACLT rodents compared with vehicle-treated ACLT controls. Halofuginone reduced collagen X (Col X), matrix metalloproteinase-13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS 5) and increased lubricin, collagen II and aggrecan. In parallel, halofuginone-attenuated uncoupled subchondral bone remodelling as defined by reduced subchondral bone tissue volume, lower trabecular pattern factor (Tb.pf) and increased thickness of subchondral bone plate compared with vehicle-treated ACLT controls. We found that halofuginone exerted protective effects in part by suppressing Th17-induced osteoclastic bone resorption, inhibiting Smad2/3-dependent TGF-β signalling to restore coupled bone remodelling and attenuating excessive angiogenesis in subchondral bone. Conclusions Halofuginone attenuates OA progression by inhibition of subchondral bone TGF-β activity and aberrant angiogenesis as a potential preventive therapy for OA.
Preclinical rheumatoid arthritis (Pre-RA) is defined as the early stage before the development of clinical RA. While cachexia is a well-known and potentially modifiable complication of RA, it is not known if such an association exists also in the Pre-RA stage. To investigate such issue, we aimed to compare the longitudinal alterations in the muscle composition and adiposity of participants with Pre-RA with the matched controls.
Abstract Ankylosing spondylitis (AS) is chronic inflammatory arthritis with a progressive fusion of axial joints. Anti-inflammatory treatments such as anti-TNF-α antibody therapy suppress inflammation but do not effectively halt the progression of spine fusion in AS patients. Here we report that the autoimmune inflammation of AS generates a microenvironment that promotes chondrogenesis in spine ligaments as the process of spine fusion. Chondrocyte differentiation was observed in the ligaments of patients with early-stage AS, and cartilage formation was followed by calcification. Moreover, a large number of giant osteoclasts were found in the inflammatory environment of ligaments and on bony surfaces of calcified cartilage. Resorption activity by these giant osteoclasts generated marrow with high levels of active TGF-β, which induced new bone formation in the ligaments. Notably, no Osterix + osteoprogenitors were found in osteoclast resorption areas, indicating uncoupled bone resorption and formation. Even at the late and maturation stages, the uncoupled osteoclast resorption in bony interspinous ligament activates TGF-β to induce the progression of ossification in AS patients. Osteoclast resorption of calcified cartilage-initiated ossification in the progression of AS is a similar pathologic process of acquired heterotopic ossification (HO). Our finding of cartilage formation in the ligaments of AS patients revealed that the pathogenesis of spinal fusion is a process of HO and explained why anti-inflammatory treatments do not slow ankylosing once there is new bone formation in spinal soft tissues. Thus, inhibition of HO formation, such as osteoclast activity, cartilage formation, or TGF-β activity could be a potential therapy for AS.
Signaling through receptors of the transforming growth factor beta (TGFbeta) superfamily is mediated by cytoplasmic Smad proteins. It has been demonstrated that Smad anchor for receptor activation (SARA) facilitates TGFbeta and activin/nodal signaling by recruiting and presenting Smad2/3 to the receptor complex. SARA does not bind Smad1 and hence does not enhance bone morphogenetic protein (BMP) signaling. Here we report for the first time that the endosome-associated FYVE-domain protein endofin acts as a Smad anchor for receptor activation in BMP signaling. We demonstrate that endofin binds Smad1 preferentially and enhances Smad1 phosphorylation and nuclear localization upon BMP stimulation. Silencing of endofin by RNAi resulted in a reduction in BMP-dependent Smad1 phosphorylation. Moreover, disruption of the membrane-anchoring FYVE motif by point mutation led to a reduction of BMP-responsive gene expression in cell culture and Xenopus ectodermal explants. Furthermore, we demonstrate that endofin contains a protein-phosphatase-binding motif, which functions to negatively modulate BMP signals through receptor dephosphorylation. Taken together, our results suggest that endofin plays an important role in both positive and negative feedback regulation of the BMP signaling pathway.
Lipoprotein receptor-related protein 6 (LRP6) plays a critical role in skeletal development and homeostasis in adults. However, the role of LRP6 in mesenchymal stem cells (MSCs), skeletal stem cells that give rise to osteoblastic lineage, is unknown. In this study, we generated mice lacking LRP6 expression specifically in nestin(+) MSCs by crossing nestin-Cre mice with LRP6(flox) mice and investigated the functional changes of bone marrow MSCs and skeletal alterations. Mice with LRP6 deletion in nestin(+) cells demonstrated reductions in body weight and body length at 1 and 3 months of age. Bone architecture measured by microCT (µCT) showed a significant reduction in bone mass in both trabecular and cortical bone of homozygous and heterozygous LRP6 mutant mice. A dramatic reduction in the numbers of osteoblasts but much less significant reduction in the numbers of osteoclasts was observed in the mutant mice. Osterix(+) osteoprogenitors and osteocalcin(+) osteoblasts significantly reduced at the secondary spongiosa area, but only moderately decreased at the primary spongiosa area in mutant mice. Bone marrow MSCs from the mutant mice showed decreased colony forming, cell viability and cell proliferation. Thus, LRP6 in bone marrow MSCs is essential for their survival and proliferation, and therefore, is a key positive regulator for bone formation during skeletal growth and remodeling.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Pain is the most prominent symptom of osteoarthritis (OA) progression. However, the relationship between pain and OA progression remains largely unknown. Here we report osteoblast secret prostaglandin E2 (PGE2) during aberrant subchondral bone remodeling induces pain and OA progression in mice. Specific deletion of the major PGE2 producing enzyme cyclooxygenase 2 (COX2) in osteoblasts or PGE2 receptor EP4 in peripheral nerve markedly ameliorates OA symptoms. Mechanistically, PGE2 sensitizes dorsal root ganglia (DRG) neurons by modifying the voltage-gated sodium channel NaV1.8, evidenced by that genetically or pharmacologically inhibiting NaV1.8 in DRG neurons can substantially attenuate OA. Moreover, drugs targeting aberrant subchondral bone remodeling also attenuates OA through rebalancing PGE2 production and NaV1.8 modification. Thus, aberrant subchondral remodeling induced NaV1.8 neuronal modification is an important player in OA and is a potential therapeutic target in multiple skeletal degenerative diseases. eLife digest Many people will suffer from joint pain as they age, particularly in their knees. The most common cause of this pain is osteoarthritis, a disease that affects a tissue inside joints called cartilage. In a healthy knee, cartilage acts as a shock absorber. It cushions the ends of bones and enables them to move smoothly against one another. But in osteoarthritis, cartilage gradually wears away. As a result, the bones within a joint rub against each other whenever a person moves. This makes activities such as running or climbing stairs painful. But how does this pain arise? Previous work has implicated cells called osteoblasts. Osteoblasts are found in the area of the bone just below the cartilage. They produce new bone tissue throughout our lives, enabling our bones to regenerate and repair. Each time we move, forces acting on the knee joint activate osteoblasts. The cells respond by releasing a key molecule called PGE2, which is a factor in pain pathways. The joints of people with osteoarthritis produce too much PGE2. But exactly how this leads to increased pain sensation has been unclear. Zhu et al. now complete this story by working out how PGE2 triggers pain. Experiments in mice reveal that PGE2 irritates the nerve fibers that carry pain signals from the knee joint to the brain. It does this by activating a channel protein called Nav1.8, which allows sodium ions through the membranes of those nerve fibers. Zhu et al. show that, in a mouse model of osteoarthritis, Nav1.8 opens too widely in response to binding of PGE2, so the nerve cells become overactive and transmit a stronger pain sensation. This means that even small movements cause intense pain signals to travel from the joints to the brain. Building on their findings, Zhu et al. developed a drug that acts directly on bone to reduce PGE2 production, and show that this drug reduces pain in mice with osteoarthritis. At present, there are no treatments that reverse the damage that occurs during osteoarthritis, but further testing will determine whether this new drug could one day relieve joint pain in patients. Introduction Subchondral bone is an integral component of the joint, absorbing compressive forces during movement (Martel-Pelletier et al., 2016). Physiological subchondral bone remodeling maintains its structural integrity and supports the overlying articular cartilage. Age or trauma (Hayami et al., 2004) related alteration of subchondral bone is a principal risk factor of osteoarthritis (OA), the most common joint disease (Berenbaum et al., 2018) characterized by cartilage destruction (Glasson et al., 2005; Kim et al., 2014a), subchondral sclerosis (Suri et al., 2007) and synovitis (Benito et al., 2005; Sellam and Berenbaum, 2010). Pain, as the major symptom for OA patients (Lane et al., 2010), often leads to physical disability and mortality in senior patients (Chen et al., 2017). Although the major local source is not clearly defined (Malfait and Schnitzer, 2013), there is evidence showing that synovitis (Sellam and Berenbaum, 2010) and subchondral bone marrow lesion (BML) are highly relevant to OA pain. BML is a fluid enriched area under magnetic resonance imaging (MRI) and is characterized as bone marrow edema, fibrosis, microfractures or trabecular pattern alterations in pathological examinations, with high relevance to abnormal bone remodeling. However, how subchondral BML induces OA pain, still remains largely unknown. We previously showed that aberrant subchondral bone remodeling in response to altered mechanical loading patterns in OA was initiated by over-activation of transforming growth factor β1 (TGF-β1). High levels of subchondral TGF-β1 signaling induces mesenchymal stem cells (MSC) clustering and leads to the uncoupling of osteoblastic bone formation, osteoclastic bone resorption and angiogenesis. Moreover, we found overactivated osteoclasts in BML aggravated OA pain by secreting the axon guidance molecule Netrin-1 to induce subchondral sensory innervation (Zhu et al., 2019). The increased sensory innervation provides a structural base for the transmission of nociceptive signals from the subchondral bone to the central nervous system. However, how these nerve fibers are activated and sensitized during OA progression remains to be elucidated. The persistent low grade of local inflammation is another hallmark of OA (Midwood et al., 2009; Tu et al., 2019). During the uncoupled bone remodeling in OA progression, a series of pro-inflammatory factors (Kapoor et al., 2011; Sellam and Berenbaum, 2010) like prostaglandin E2 (PGE2) (Chen et al., 2019), interleukin-1β (IL-1β), interleukin-6 (IL-6) are released into the subchondral bone area (Massicotte et al., 2002). We previously showed that PGE2 was a crucial factor in both pain sensation and bone metabolism (Tu et al., 2019). However, the main source remains largely unknown. We newly discovered a feedback mechanism on sensory nerve regulation of bone mass. PGE2 concentration is inversely related to bone mass and sensory nerves monitors bone density by responding to the concentration of PGE2 in bone (Chen et al., 2019). We found that when bone mass is decreased, the enzymatic activity of cyclooxygenase 2 (Cox2) in osteoblastic lineage cells was significantly increased and thus catalyzes more arachidonic acid into PGE2. At the early stage of OA, the bone density is temporarily decreased, which resembles the low bone density as seen in osteoporosis. Currently, there is still lack of information whether the increased PGE2 in OA subchondral bone is also attributed to the increased Cox2 activity in osteoblastic cells in response to low bone mass. PGE2 functions as an inflammatory mediator and a neuromodulator that alters neuronal excitability (Samad et al., 2001). In the four types of G-protein-coupled EP receptors (EP1-EP4) that mediate the functions of PGE2, EP4 receptor is considered as the primary mediator of PGE2- evoked inflammatory pain hypersensitivity and sensitization of sensory neurons (Boyd et al., 2011; Chen et al., 2008; Lin et al., 2006; McCoy et al., 2002; Nakao et al., 2007; Southall and Vasko, 2001; Taylor-Clark et al., 2008), as evidenced by that the specific EP4 receptor antagonists could reduce acute and chronic pain (Nakao et al., 2007), including OA pain (Abdel-Magid, 2014). Increased neuronal excitability contributes to the generation of hypersensitivity in various types of chronic pain (Kuner, 2010; Malfait and Schnitzer, 2013). PGE2 has been shown to potentiate several ion channels in neurons to enhance neuronal excitability (Funk, 2001). Voltage-gated sodium channel (NaV), a member of the tetrodotoxin-resistant sodium channel (TTX-R), is mainly expressed in small- and medium-sized Dorsal root ganglion (DRG) neurons and their fibers. The NaV is responsible for initiating and propagating electrical signal transmission by inducing Na+ influx to start action potential firing. PGE2 has been shown to modulate the sodium current of the TTX-R in DRG neurons and promote Nav1.8 trafficking to the cell surface (England et al., 1996; Liu et al., 2010). Therefore, the PGE2 induced neuronal hypersensitivity is likely to be mediated by the Nav 1.8 during OA progression. Among the 9 subtypes of NaVs (NaV1.1–1.9), NaV1.8 (Akopian et al., 1996) is the main drug target due to its highly relevant to pain signal transmission, and restricted distribution in primary nociceptive neurons (Akopian et al., 1999; Julius and Basbaum, 2001). Gain of function mutations in human in the promoter region of NaV1.8 directly induces pain hypersensitivity (Duan et al., 2018). Interestingly, animals lacking NaV1.8 display significant lower mechanical pain sensitivity with modest changes in heat or innocuous touch sensitivities (Akopian et al., 1999; Basbaum et al., 2009). This specificity of NaV1.8 in transducing mechanical pain signals makes it highly possible in the participation of mechanical allodynia in OA. Moreover, post-transcriptional modifications of NaV1.8 including phosphorylation (Gold et al., 1998; Hudmon et al., 2008; Wu et al., 2012) and methylglyoxalation (Bierhaus et al., 2012) further regulate its activity. A recent study demonstrated that inhibition of the expression of NaVs in nociceptive neurons was effective in OA pain alleviation (Miller et al., 2017), with the detailed molecular mechanism remained to be clarified (Strickland et al., 2008). In this study, we take the initiative to show that aberrant subchondral bone remodeling contributes to neuronal hypersensitivity during OA progression. Excessive PGE2 is synthesized by osteoblastic lineage cells in response to the low bone density at the early stage of OA. Excessive PGE2 sensitize sensory fibers innervates subchondral bone by upregulating the expression of sodium channel NaV1.8 in both subchondral bone nerve fibers and DRG neuron body, which contributes to peripheral mechanical allodynia during OA progression. Therefore, we developed a small molecule conjugate by linking the TGFβ type receptor 1 (Tβ1R) inhibitor (LY-2109761) and alendronate (Aln) (Hayami et al., 2004) to achieve bone-targeted delivery. We used this conjugate (Aln-Ly) as a proof of concept drug to test whether reversing the aberrant bone remodeling by synergistically inhibiting osteoclast bone resorption and the excessive TGF-β activity can substantially reduce the PGE2 production and subsequent mechanical hypersensitivity that generated in OA subchondral bone. Results NaV1.8 was modified in the subchondral bone and mediate OA progression To identify the primary voltage-gated sodium channel in subchondral sensory fibers that responsible for mechanical hypersensitivity during OA progression, we tested the expression levels of different sensory related sodium channel NaVs in OA mice post anterior cruciate ligament transection (ACLT). The transcription levels of mRNAs that encode NaVs in DRG including NaV1.1, NaV1.2, NaV1.3, NaV1.6, NaV1.7, NaV1.8, NaV1.9 were measured by qPCR using mRNA isolated from mouse ipsilateral L3-5 DRGs one month post-ACLT or sham surgery. Compares to the sham-operated group, the expression of mRNA encoding NaV1.8 increased 2.5 folds in the ACLT group as the highest upregulation among all the NaVs. The mRNAs encoding NaV1.7 and NaV1.9 showed moderate upregulation in the ACLT group relative to that of the Sham group while the changes of NaV1.1 NaV1.2 NaV1.3 or NaV1.6 were not detected (Figure 1a). Therefore, we further investigated NaV1.8 protein expression in the immune-histological analysis of OA subchondral bone. The intensity of NaV1.8 immunofluorescence in subchondral bone was also elevated about 2 to 3 folds in OA mice compared to sham- operated mice one- or two-months post-surgery (Figure 1b and c). To examine whether the increase of NaV1.8 expression is limited to in a certain subtype(s) of the DRG neuron that innervates subchondral bone in OA mice, NaV1.8 was co-stained with different markers for sensory nerve subtypes based on the current classification of DRG neurons (Usoskin et al., 2015). The expression rate of NaV1.8 in total nerve fibers (labeled by pan neuron marker PGP9.5) innervated in subchondral bone significantly increased post-ACLT (Figure 1—figure supplement 1a,f). Moreover, the elevated NaV1.8 expression was highly co-localized with the peptidergic nociceptor marked by calcitonin gene-related peptide (CGRP) (Brain et al., 1985; Figure 1—figure supplement 1b,f) and mechanosensitive low-threshold mechanoceptors (labeled by PIEZO2) (Eijkelkamp et al., 2013). The expression of NaV1.8 was also slightly elevated in the synovium (Figure 1—figure supplement 1f 1 hr, j). Both western blot analysis (Figure 1f) and immunostaining (Figure 1—figure supplement 1f 1 g, i) of ipsilateral lumbar 3–5 DRG confirmed the upregulation of NaV1.8 expression at the DRG level. We then further validated whether DRG neurons with upregulated NaV1.8 expression directly innervates fibers in the subchondral bone. We injected a neurophilic fluorescent dye (DiI) into the subchondral bone to label the distal nerve fibers (Ferreira-Gomes et al., 2010). We found that the NaV1.8+ neurons labeled by DiI significantly increased in the DRG neurons of OA rats relative to sham-operated rats (Figure 1g,h), indicating that DiI was transported into DRG neurons by the sensory fibers innervated subchondral bone in a retrograde manner. These results suggest that the expression of pain-related sodium channel NaV1.8 is upregulated in DRG neurons and their axons that innervate subchondral bone during progression. We then examined the association between NaV1.8 neuronal activity and OA pain. Von Frey test showed that the hind paw withdrawal threshold (HPWT) dropped nearly 60% and maintained at this level throughout the two month-period post ACLT relative to the sham-operated group, suggesting that development of mechanical allodynia in OA mice (Figure 1g; Chen et al., 2017). To assess the potential role of NaV1.8 in DRG neuronal excitability, we used an in vivo DRG imaging in PirtGCaMP3fl/-mice that we recently developed. In this genetically targeted mice, genetic-encoded Ca2+ indicator GCaMP3 is specifically expressed in >95% of all DRG neurons under the control of the Pirt promoter. In the PirtGCaMP3fl/-mice, the excitability of the nociceptive neurons in DRG can be visualized by fluorescence signals of calcium influx. The number of excited DRG neurons ipsilateral to the surgery significantly increased in OA mice compared to sham-operated mice, and importantly, administration of NaV1.8 inhibitor (A-803467)(Jarvis et al., 2007) blunted the signal in DRG (Figure 1h and i). To validate the excitation of DRG neurons related to NaV1.8, we performed patch-clamp in the DRG neurons that were isolated from mice that underwent ACLT or sham surgery. The action potential number and NaV1.8 current density significantly elevated in ACLT mice relative to sham-operated mice, and the elevations were blocked by A-803467 (Figure 1j–l, Figure 1—figure supplement 1f 1 k and l). Thus, the activation of NaV1.8 mediates OA pain related DRG neuron hypersensitivity. Figure 1 with 1 supplement see all Download asset Open asset Nav1.8 modification after mice model of OA. (a) Heatmap of relative expression levels of NaV channels in ipsilateral L3-L5 DRGs after sham or ACLT surgery. (b, c) Immunostaining of NaV1.8+ (green) nerve fibers (b) and statistical analysis (c) in mouse tibial subchondral bone after sham or ACLT surgery at 1 m and 2 m. Scale bars, 20 μm, n = 6 per group. (d, e) Immunostaining of NaV1.8+ (green) nerve fibers (d) and statistical analysis (e) in ipsilateral sciatic nerve after sham or ACLT surgery at 1 m. Scale bars, 40 μm, n = 6 per group. (f) Western blots of NaV1.8 in mouse ipsilateral L3-5 DRGs 1 month post sham or ACLT surgery. the experiment was repeated three times and a representative result was chosen. (g, h) Retrograde tracing of Nav1.8 (green) and DiI (red) and DAPI (blue) double-labeled neurons (g) and percentage of double labeled neurons (h) in ipsilateral L4 DRG of rat after sham or ACLT surgery in 3 m. Scale bar, 80 μm. n = 6 per group. **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (c, k, l), unpaired Student’s t test (e an i) and all data are shown as scattered plots with means ± standard deviations. (h, i) Representative photomicrographs (h) and statistically analysis of activated neurons (i) in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging treated before or after A803467 1 month post sham or ACLT surgery. n = 6 per group. (j–l) Representative traces of Aps (j upper), maximal current density (j lower), statistical analysis of AP numbers (k) and NaV1.8 currents (l) of DRG 1 month post sham or ACLT. **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (c, k, l), unpaired Student’s t test (e an i) and all data are shown as scattered plots with means ± standard deviations. Figure 1—source data 1 Raw data of Navs QPCR, subchondral Nav1.8 fiber density, Retrograde tracing, Von Frey tests, GcAMP3 imaging, and electrophysiological recordings. https://cdn.elifesciences.org/articles/57656/elife-57656-fig1-data1-v2.xlsx Download elife-57656-fig1-data1-v2.xlsx Figure 1—source data 2 Full scan of western blots in Figure 1f. https://cdn.elifesciences.org/articles/57656/elife-57656-fig1-data2-v2.pdf Download elife-57656-fig1-data2-v2.pdf Excessive PGE2 secreted by osteoblasts modifies NaV1.8 for OA We then examined the mechanism of upregulation of NaV1.8 expression during OA progression. To examine whether excessive PGE2 contributes to the upregulation of NaV1.8, we firstly performed immunostaining of cyclooxygenase 2 (Cox2) in subchondral bone sections. Cox2 expression was significantly increased in subchondral bone and primarily in osteocalcin positive osteoblastic cells post ACLT mice compared with sham-operated mice (Figure 2a and b). Consistently, PGE2 concentration in subchondral bone increased about three times in OA mice relative to sham-operated mice (Figure 2c, Figure 2—figure supplement 1a–d). To examine if elevated PGE2 upregulates the expression of NaV1.8 for the mechanical allodynia in OA, we generated osteoblast specific Cox2 deficient mice (Cox2Oc-/- mice) by crossbreeding Cox2fl/fl mice with Bglap-Cre mice. PGE2 concentration in subchondral bone was significantly lower in Bglap-Cre::Cox2fl/fl mice compared with Cox2fl/fl mice post ACLT (Figure 2h). Notably, the NaV1.8 immunofluorescence intensity in subchondral bone immunostaining was also significantly reduced in Bglap-Cre::Cox2fl/fl mice compared with Cox2fl/fl mice (Figure 2d and i). In addition, the number of NaV1.8+ neurons in DRG was also significantly decreased (Figure 2e and j) in the Bglap-Cre::Cox2fl/fl mice relative to Cox2fl/fl mice. We then investigated whether a decrease of PGE2 alleviates OA pain. We crossed Bglap-Cre::Cox2fl/fl mice with Pirt GCaMP3fl/- mice to measure the DRG neuronal excitability in Bglap-Cre::Cox2fl/fl::Pirt GCaMP3fl/- mice post ACLT. Pirt-GCaMP3 DRG imaging showed that the number of excited neurons was significantly reduced in Bglap-Cre::Cox2fl/fl::PirtGCaMP3fl/- mice compared with Cox2fl/fl::Pirt GCaMP3fl/- mice post ACLT (Figure 2f and k). Moreover, the single neuron excitability in ipsilateral L4 DRG was functionally tested by whole cell patch clamp electrophysiology. The whole cell current clamp revealed that the action potential firing number was significantly decreased in Bglap-Cre::Cox2fl/fl mice compared with Cox2fl/fl mice after ACLT (Figure 2g and l). Concurrently, the NaV1.8 current density was reduced for about 40% recorded by the whole-cell voltage-clamp (Figure 2g and m). The mechanical allodynia was simultaneously attenuated in Bglap-Cre::Cox2fl/flmice relative to Cox2fl/fl mice as revealed by the Von Frey behavior test (Figure 2n). Catwalk analysis also showed that the Maximal Contact At and Maximal Intensity of ipsilateral hind paw was significantly higher in Bglap-Cre::Cox2fl/fl mice compared with Cox2fl/fl mice post ACLT (Figure 2o). Thus, PGE2 derived from osteoblastic cells stimulates the pain hypersensitivity in OA mice likely by upregulating of NaV1.8 in subchondral nociceptive neurons. Figure 2 with 1 supplement see all Download asset Open asset Decreased NaV1.8 expression and ameliorated OA progression in Cox2:OCN cKO ACLT mice. (a,b) Representative pictures (a) and statistical analysis (b) of OCN and Cox2 co-stained cells of murine tibial subchondral bone after sham or ACLT surgery and 1 m. Scale bars, 50 μm (left) and 10 μm (right), n = 6 per group. (c) Relative concentration of subchondral PGE2 compared with total protein concentration before and after ACLT. (e–g) Nav1.8 (green) immunostaining in subchondral bone (d), NeuN (red), NaV1.8 (green) and DAPI (blue) co-immunostaining in ipsilateral L4 DRG (e), Activated neurons in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging (f) and AP traces and NaV currents (g) after sham or ACLT surgery at 1 m. Scale bars, 20 μm (h), 100 μm (e, f). (h–o) Statistical analysis of subchondral PGE2 concentration (h), NaV1.8 immunofluorescence signal in subchondral bone (i), number of NeuN, Nav1.8 co-immunostained neurons in ipsilateral L4 DRG (j), number of activated neurons in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging (k), AP traces (l) and NaV currents (m), Catwalk gait analysis (n) and left HPWT (o) after sham or ACLT surgery. n = 6 per group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (h) or unpaired Student’s t test (b, c, h–m and o), and all data are shown as scattered plots with means ± standard deviations. Figure 2—source data 1 Raw data of OCN Cox2 costaining, subchondral PGE2, GcAMP3 imaging, NeuN Nav1.8 merged neurons, electrophysiological recordings, Von Frey tests and catwalk analysis. https://cdn.elifesciences.org/articles/57656/elife-57656-fig2-data1-v2.xlsx Download elife-57656-fig2-data1-v2.xlsx PGE2 signals through the EP4 receptor to sensitize sensory nerves in OA subchondral bone To examine whether EP4 at sensory neurons is the primary receptor that propagates PGE2 signals in upregulating NaV1.8and OA pain, we specifically knocked out EP4, the skeletal pain related receptor for PGE2 (Yoshida et al., 2002), in peripheral sensory nerves by crossbreeding Advillin-Cre (Avil-Cre) (Zurborg et al., 2011) mice with Ptger4 fl/fl mice (Ptger4 is the gene that encodes EP4 receptor). Consistently with Bglap-Cre::Cox2fl/fl mice, the intensity of NaV1.8 immunofluorescence was significantly reduced in Avil-Cre::Ptger4 fl/fl mice compared with Ptger4 fl/fl mice post-ACLT (Figure 3a,d, Figure 2—figure supplement 1e–h). We then further confirm this finding in DRG neurons that cultured in PGE2. We found that NaV1.8 protein expression was significantly reduced by siRNA against EP4 in western blot analysis of levels from the cell lysates. Knocking-down the expression of EP1-EP3 did not have a significant effect on Nav1.8 expression (Figure 2—figure supplement 1i). To determine the effect of conditional deletion of EP4 on DRG neuronal excitability, we generated Avil-Cre::Ptger4 fl/fl::Pirt - GCaMP3fl/- and Ptger4 fl/fl::Pirt GCaMP3fl/- ACLT mice. In vivo ipsilateral L4 DRG Pirt GCaMP3 imaging demonstrated significantly dampened excitability in the Ptger4 fl/fl:: Pirt - GCaMP3fl/- mice relative to the control group (Figure 3b and e). The patch-clamp analysis further revealed that the DRG neuronal hypersensitivity and NaV1.8 currents were significantly reduced in Avil-Cre::Ptger4 fl/fl mice relative to Ptger4 fl/fl mice post-ACLT (Figure 3c,f,g). Moreover, both Von Frey test and catwalk analysis showed attenuation of OA pain when EP4 is conditionally deleted in sensory neurons (Figure 3h and i). Thus, the EP4 receptor expressed in DRG neurons is responsible for the propagation of subchondral PGE2-induced upregulation of NaV1.8 and neuronal excitability in OA mice. Figure 3 Download asset Open asset Decreased NaV1.8 expression and ameliorated mechanical allodynia in Avil-Cre::Ptger4fl/fl ACLT mice. (a, b) NaV1.8 immunostaining in subchondral bone (a), Activated neurons in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging (b) after sham or ACLT surgery at 1 m. Scale bars, 20 μm (a), 100 μm (b). (c) Representative traces of action potentials (upper) and Nav1.8 currents (lower) of ipsilateral L3-5 DRG neurons after sham or ACLT surgery at 1 m. (d–i) Statistical analysis of Nav1.8 immunofluorescence signal in subchondral bone (d), number of activated neurons in ipsilateral L4 DRG using in vivo Pirt-GCaMP3 imaging (e), AP traces (f), max Nav1.8 current density (g), catwalk gait analysis (h) and left hindpaw PWT (i) after sham or ACLT surgery. n = 6 per group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (i) or unpaired Student’s t test (d–h), and all data are shown as scattered plots with means ± standard deviations. Figure 3—source data 1 Raw data of subchondral Nav1.8 fiber density, Von Frey tests, catwalk analysis, GcAMP3 imaging, and electrophysiological recordings. https://cdn.elifesciences.org/articles/57656/elife-57656-fig3-data1-v2.xlsx Download elife-57656-fig3-data1-v2.xlsx PGE2 stimulates NaV1.8 transcription by inducing the binding of pCREB to NaV1.8 promoter To investigate the mechanism of PGE2 stimulated upregulation of NaV1.8 expression, RT-qPCR was performed with mRNA isolated from primary DRG neurons treated with PGE2. The result showed that NaV1.8 transcription levels were significantly elevated at 6 and 12 hr after incubation with PGE2 (Figure 4a). Moreover, PGE2 stimulated phosphorylation of protein kinase A (PKA) (Gold et al., 1998) and cAMP response element-binding protein (Creb1) (Lonze and Ginty, 2002; Figure 4b). Notably, the effect of PGE2 and Forskolin, a cAMP stimulant in the upregulation of NaV1.8 protein expression was dampened by Creb1 inhibitor 666–15 (Figure 4c), indicating PGE2 stimulates Nav1.8 expression through the PKA-Creb1 signaling pathway. Consistently, the neuronal excitability and NaV1.8 current density stimulated by PGE2 was abolished by the application of PKA inhibitor (PKI) or CREB inhibitor 666–15 (Figure 4d–f). Co-immunofluorescence staining further demonstrated that PKA levels significantly increased in NaV1.8 positive DRG neurons in ACLT mice compared with sham-operated mice (Figure 4g and h). To examine the mechanism of PGE2-induced NaV1.8 transcription, we performed chromatin immunoprecipitation (ChIP) assay with three potential pCreb1-binding elements in the NaV1.8 promoter. ChIP assay revealed that pCreb1 binds to the NaV1.8 promoter at binding site two to stimulate transcription of NaV1.8 gene (Figure 4i–k). Taken together, our findings reveal that PGE2 induces transcription of NaV1.8 by stimulation phosphorylation PKA and pCreb1, which directly binds to NaV1.8 promoter. Figure 4 Download asset Open asset PGE2 upregulates NaV1.8 through PKA signaling. (a) RT-QPCR analysis of Nav1.8 in cultured lumbar DRG neurons treated with PGE2 (1 μM) for 2–12 hr. n = 6 per group. (b) Representative of western blots of PKA-c, CREB and p-CREB in cultured lumbar DRG neurons treated with PGE2 (1 μM) for 40–160 min. (c) Representative of western blots of the Nav1.8 in cultured lumbar DRG neurons treated with PGE2 (1 μM), forskolin (10 μM), or 666–15 (1 μM) for 6 hr. Representative traces of action potentials (d, upper) and Nav1.8 currents (d, lower) and statistical analysis of maximal Nav1.8 current density (f) of cultured lumbar DRG neurons after sham or ACLT surgery at 1 m. n = 6 per group. (e, f) Co-immunostaining of NeuN and Nav1.8 (g) and statistical analysis of merged cell numbers (h) in ipsilateral L4 DRGs after sham or ACLT surgery at 1 m. n = 6 per group.(i–k) ChIP experiment showing putative primers (i), PCR (j) and gel running results (k) of NaV1.8 promoter, the experiments were repeated three times. n.s, non significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the sham-operated group at different time points. Statistical significance was determined by multifactorial ANOVA WITH BONFERRONI POST HOC TEST (a, e and f), unpaired Student’s t test (h), all data are shown as scattered plots with means ± standard deviations. Figure 4—source data 1 Raw data of Navs QPCR, subchondral Nav1.8 fiber density, Retrograde tracing, Von Frey tests, GcAMP3 imaging, and electrophysiological recordings. https://cdn.elifesciences.org/articles/57656/elife-57656-fig4-data1-v2.xlsx Download elife-57656-fig4-data1-v2.xlsx Figure 4—source data 2 Full scan of western blots in Figure 4b. https://cdn.elifesciences.org/articles/57656/elife-57656-fig4-data2-v2.pdf Download elife-57656-fig4-data2-v2.pdf Figure 4—source data 3 Full scan of western blots in Figure 4c. https://cdn.elifesciences.org/articles/57656/elife-57656-fig4-data3-v2.pdf Download elife-57656-fig4-data3-v2.pdf Figure 4—source data 4 Full scan of western blots in Figure 4k. https://cdn
Abstract Background TGF-β resistance often develops in breast cancer cells that in turn overproduce this cytokine to create a local immunosuppressive environment that fosters tumor growth and exacerbates the invasive and metastatic behavior of the tumor cells themselves. Smads-mediated cross-talk with the estrogen receptor has been implied to play an important role in development and/or progression of breast cancer. We investigated how TGF-β regulates ERα-induced gene transcription and potential mechanisms of frequent TGF-β resistance in breast cancer. Methods Effect of TGF-β on ERα-mediated gene transcription was investigated in breast cancer cell lines using transient transfection, real-time PCR, sequential DNA precipitation, and small interfering RNA assays. The expression of Smads on both human breast cancer cell lines and ERα-positive human breast cancer tissue was evaluated by immunofluorescence and immunohistochemical assays. Results A complex of Smad3/4 mediates TGF-β inhibition of ERα-mediated estrogenic activity of gene transcription in breast cancer cells, and Smad4 is essential and sufficient for such repression. Either overexpression of Smad3 or inhibition of Smad4 leads to the "switch" of TGF-β from a repressor to an activator. Down-regulation and abnormal cellular distribution of Smad4 were associated with some ERα-positive infiltrating human breast carcinoma. There appears a dynamic change of Smad4 expression from benign breast ductal tissue to infiltrating ductal carcinoma. Conclusion These results suggest that aberrant expression of Smad4 or disruption of Smad4 activity lead to the loss of TGF-β suppression of ERα transactivity in breast cancer cells.
Spinal pain affects individuals of all ages and is the most common musculoskeletal problem globally. Its clinical management remains a challenge as the underlying mechanisms leading to it are still unclear. Here, we report that significantly increased numbers of senescent osteoclasts (SnOCs) are observed in mouse models of spinal hypersensitivity, like lumbar spine instability (LSI) or aging, compared to controls. The larger population of SnOCs is associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. We show that deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation and H-type vessel growth in the endplate. We also show that there is significantly increased SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. These findings suggest that pharmacological elimination of SnOCs may be a potent therapy to treat spinal pain.
Spinal pain affects individuals of all ages and is the most common musculoskeletal problem globally. Its clinical management remains a challenge as the underlying mechanisms leading to it are still unclear. Here, we report that significantly increased numbers of senescent osteoclasts (SnOCs) are observed in mouse models of spinal hypersensitivity, like lumbar spine instability (LSI) or aging, compared to controls. The larger population of SnOCs is associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. We show that deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation and H-type vessel growth in the endplate. We also show that there is significantly increased SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. These findings suggest that pharmacological elimination of SnOCs may be a potent therapy to treat spinal pain.
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