Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through αE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin upon binding to the E-cadherin•β-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in αE-catenin when bound to F-actin. Comparisons of wild-type αE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal β-catenin-binding and the middle (M) vinculin-binding domain of αE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length αE-catenin, and identify the M domain as a key transmitter of conformational changes. In these assemblies, the extracellular portion of classical cadherins mediates homophilic cell-cell adhesion. In epithelial tissues, the cadherin intracellular domain binds to β-catenin, which in turn associates with the F-actin-binding protein αE-catenin  and moderate (8 pN) forces12. Furthermore, at forces of approximately 5 pN, αE-catenin recruits vinculin, which is thought to reinforce the AJ through its own actin-binding activity17  are key cell-cell adhesion complexes that regulate tissue morphogenesis, differentiation and wound healing, and their disruption is associated with cancer metastasisnin Fig. 11. Notaby17 Fig. . This fuI and NII) that both binds β-catenin and mediates homodimerization; a middle (M) domain consisting of three four-helix bundles  that harbor binding sites for vinculin and other protein partners; and a C-terminal, five-helix bundle termed the actin-binding domain (ABD)26  simulations, combined with mutational analysis, have identified salt bridge interactions within the M domain whose dynamics are thought to be associated with regulated vinculin binding34.The structure of αE-catenin comprises a series of α-helical bundles organized into three main regions: an N-terminal (N) domain that consists of two four-helix bundles (N)26 Fig. . StructuDespite the advances outlined above, the structural responses of full-length αE-catenin to its binding partners and force are poorly understood. Little is known about F-actin binding by αE-catenin, including the molecular mechanisms by which cadherin-β-catenin inhibits, and force promotes, binding of αE-catenin to F-actin. Here, we examine the conformational dynamics and inter-domain coupling of αE-catenin associated with its binding to cadherin•β-catenin, F-actin, and vinculin by measuring changes in native cysteine accessibility in solution. We developed a novel method to directly probe the effect of mechanical force on αE-catenin by examining changes in cysteine accessibility under physiologically relevant levels of mechanical load induced by fluid shear. Combined with structural and thermodynamic data, these experiments provide direct experimental data for structural changes associated with allosteric regulation of αE-catenin.18, and αE-catenin 82–906 at 3.7 Å (PDB 4IGG) in which the ABDs in each protomer were ordered, but adopted different positions with respect to the rest of the molecule26. Small angle x-ray scattering (SAXS) analysis of the full-length protein suggested a more extended conformation than that observed in the 4IGG structure35. The absence of the N-terminal 81 residues, and/or that the crystals were dehydrated to improve resolution of the 4IGG structure, raised the possibility that the positions of the ABDs in 4IGG do not represent the solution structure of the αE-catenin dimer. Therefore, we determined the crystal structure of the αE-catenin 82–883 dimer at 4.0 Å resolution without dehydration, and also examined its structure in solution by SAXS. The ABDs in αE-catenin 82–883 crystals were disordered, and the protein is extended in solution N18, and e.g., αE-catenin under load).We used site-specific thiol labeling followed by mass spectrometry to determine changes in native cysteine accessibility in isolated wild-type and R551A mutant αE-catenin, in its complexes with E-cadherin and β-catenin, F-actin and vinculin, and under force when bound to F-actin. For all experiments in solution, our strategy was to label pre-formed, saturated complexes, so that any changes in labeling between different complexes reflect conformational differences between them. Note that although these complexes are saturated at equilibrium in these experiments, we cannot exclude that labeling of a small fraction of dissociated proteins at equilibrium may occur. More significantly, for complexes of αE-catenin with F-actin immobilized on glass (see below), saturating levels of F-actin were mixed with αE-catenin at the same time as the label, so effects of the label on αE-catenin interactions with its partners cannot be excluded. However, even if Cys modifications were to alter the structure, differences in reactivity must reflect changes in structure or dynamics. Thus, direct pairwise comparisons of cysteine labeling patterns under different conditions provides a means to derive meaningful insight into differences in αE-catenin conformation under conditions that are otherwise difficult to access  The relatively small size of mBBr allows it to probe modest changes in conformation such as small rearrangements of interdomain interfaces expected from prior structural and computer simulation studies.We chose the fluorescent thiol-reactive probe monobromobimane (mBBr), which is roughly the size of a tryptophan side chain, in part for a possible future application to directly label αE-catenin in cells, as mBBr can cross membranesI, which have limited solvent accessibility in crystal structures, label strongly; this region is also proteolytically sensitive37, indicating that MI is relatively unstable and flexible. The mass spectrometry data were not as reliable in the ABD due to the proximity of cysteines in the digested peptides, which made determination of labeling efficiency at a given site challenging.Murine αE-catenin has 12 cysteine residues, which the crystal structures reveal have variable levels of solvent exposure Fig. . We estaII and MII bundles , C228 in NII becomes significantly more exposed in αE-catenin  and cysteines 720 and 787 in the ABD when WT αE-catenin is bound to E-cadherin•β-catenin. Although the available structural data do not provide an obvious explanation for these results, they do provide experimental evidence for coupling between the β-catenin binding N domain and the M and F-actin-binding domains of αE-catenin  binds to actin filaments. A recent SAXS/SANS study showed that when bound to F-actin, the αE-catenin ABD is separated from the remainder of the protein, tethered by the long loop that connects the M domain to the ABD787 Fig. , indicat787 Fig. . The one18. A charge reversal mutant at this site, R551E, as well as other mutations designed to disrupt the salt bridge network, likewise enhance binding to vinculin30. Using isothermal titration calorimetry (ITC), we found that full-length R551A bound to the αE-catenin binding D1 domain of vinculin with essentially the same dissociation constant as the isolated MI-MII fragment of the wild-type αE-catenin (KD = 4 nM), rather than the 475x weaker binding of the full-length wild-type protein (Table D of 26 nM (Table I), C438 (MII) and C461 (MII) was observed in isolated R551A vs. wild type αE-catenin . LikewinM Table . SAXS menin KD =  nM, rathnin KD =  nM, rathI bundle22, indicating that the N and M domains are thermodynamically coupled. As in the wild-type case .Current high-resolution structural methods such as x-ray crystallography cannot examine molecules under tension, so we developed a method to apply force to αE-catenin in solution while simultaneously probing cysteine accessibility. We used a cone and plate rheometering Fig. . In thisA, where τ is the shear stress of the solution and A is the area occupied by the protein39. The shear stress depends upon the shear rate of the moving cone and the viscosity of the liquid solution39. To achieve a sufficient viscosity, 50% glycerol was present in the solution. Using a shear rate of 6,000 s−1, the measured viscosity was ~0.02 N s m−2, which is approximately 20 times that of pure water and closer to the viscosity of the cytoplasm40. These parameters give a shear stress τ = 120 Pa. To achieve physiologically meaningful forces, we added actin filaments at a concentration that saturates αE-catenin, which gave an average area of 0.039 μm2 for the αE-catenin•F-actin complex . With these values for the shear stress and area, we estimate that a force of ~4.7 pN is applied to αE-catenin bound to a single actin filament of average length. Complexes with longer filaments experience larger forces linearly proportional to filament length; in vitro polymerized filaments follow an approximately exponential distribution of lengths up to about 20 μm, so a small fraction of αE-catenin•F-actin complexes could experience forces significantly higher than 4.7 pN. Conversely, a significant fraction of filaments is shorter than 4.9 μm, with correspondingly smaller forces experienced by complexes with αE-catenin. Despite these uncertainties, given that the force regime experienced by that αE-catenin in AJs was recently determined to be 2–8 pN12, and that force promotes strong binding to vinculin at 4.5 pN21, the forces applied here are in a physiologically relevant range.The force applied to the immobilized molecules can be estimated from the equation F = τ x I), C526 (MIII), and C767 and C772 in the ABD. These changes might indicate some sensitivity of the dynamics of these domains to solvent properties. We next assessed whether the immobilization on glass required for the cone-and-plate rheometer measurements affects accessibility  and C526 (MIII) were labeled more in the presence of shear force  C438 is buried at the interface between MI and MIII domains that is stabilized by the E277-R451 salt bridge  and D536 (MIII) ruptured just before separation of MI from MIII . These results indicate that force has effects beyond simply disrupting the salt bridges, likely by promoting larger conformational shifts such as whole domain movements.12. Vinculin binds to αE-catenin weakly in solution, but removal of the MIII domain, also known as the “adhesion modulation domain”41, as well as the first 56 residues of αE-catenin, results in strong vinculin binding22. Force enhances binding to vinculin20, likely due to formation of sequential structural intermediates in the M domain that facilitate exposure of the vinculin-binding site34.By connecting the cadherin•β-catenin complex to F-actin, αE-catenin can sense and transmit internally generated and externally applied forces at the adherens junction. The interactions of αE-catenin are allosterically modulated by binding to its protein partners and by mechanical load. Binding of the cadherin•β-catenin complex to αE-catenin weakens association of αE-catenin with F-actin in solution, but the linkage of the cadherin•catenin complex with F-actin is strengthened by forceII and in M domains upon binding of αE-catenin to the E-cadherin•β-catenin complex  under shear suggests that the conformational equilibrium of the MI bundle does not change in response to these low-medium forces. On the other hand, the labeling of C337 changed in the R551A mutant, indicating that the mutation affects the conformational equilibrium; this was also manifest in increased labeling at C438  are well-established methods that can quantitatively apply force and detect the stress response of various mechanical anchorsEscherichia coli BL21 cells. Cells were grown to OD600 = 0.8 at 37 °C and induced overnight at 18 °C with 0.5 mM isopropyl 1-thio-β-D-galactopyranoside. The proteins were purified on a glutathione–agarose GST affinity column. After equilibration into 20 mM Tris pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 10% glycerol αE-catenin was cleaved from the GST-tag on the column by overnight incubation with TEV at 4 °C. The eluate from the GST column was purified by anion exchange chromatography  in 20 mM Tris pH 8.0, 1 mM DTT with a 0 to 500 mM NaCl gradient.Murine αE-catenin constructs were cloned into a pGEX-2T vector that was modified to introduce a tobacco etch virus protease (TEV) cleavage site after the N-terminal glutathione-S-transferase (GST) affinity tag. Point mutations were introduced by site directed mutagenesis. For crystallography, the full-length construct was truncated to residues 82–883, and Cys116 of the full-length sequence was mutated to serine. The constructs were expressed in −1. For ITC experiments, all proteins were prepared with 20 mM HEPES, pH 8.0, 150 mM NaCl and 1 mM DTT. This same buffer was used for the SAXS experiments on αE-catenin 82–883. To prepare αE-catenin for SEC-SAXS analysis, the size exclusion column was run in phosphate buffered saline, 1 mM DTT and 1% glycerol; pooled monomer fractions were concentrated shortly before data collection to 100 μM. Murine β-catenin, chicken vinculin, vinculin D1 domain (residues 1–259) and the cytoplasmic domain of E-cadherin were purified as described22.Following anion exchange chromatography, αE-catenin constructs were further purified by size exclusion chromatography  in different buffers depending on the experiment. For crystallization, the protein was purified in 20 mM Tris pH 8.0, 150 mM NaCl and 1 mM DTT, and concentrated to 10–20 mg mlEscherichia coli BL21 cells and purified as described previously47. Monomeric αE-catenin fractions from preparative size exclusion chromatography were used immediately after purification for the solution labeling experiments and for surface immobilization. G-actin was purified from rabbit muscle acetone powder 48, flash frozen and stored at −80 °C in G-actin buffer 5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2, and 0.2 mM ATP). Thawed 20 μM G-actin was polymerized by addition of 10x F-actin buffer , followed by a 1 hour incubation at room temperature in the presence of 22 μM phalloidin .For cysteine labeling experiments, wild-type and R551A αE-catenin were expressed using the pGEX-2T vector described above but modified to contain an 8-His tag following the TEV cleavage site. These were expressed in D of 1 μM49. Labeling was stopped by adding reduced glutathione to a final concentration of 5 mM for 1 hour at 4 °C. The labeled protein or protein complexes were subjected to non-reducing SDS-polyacrylamide gel electrophoresis. αE-catenin bands were excised from the gel and submitted for mass spectrometry analysis at the Stanford Mass Spectrometry Facility.Ternary E-cadherin•β-catenin•αE-catenin complex, and E-cadherin•β-catenin•αE-catenin R551A mutant complex bound to D1 were formed by mixing the purified components and then purified by size exclusion chromatography ; for actin complexes, αE-catenin was mixed with saturating amounts of F-actin. All labeling of the proteins in solution was performed after complex formation, except for the experiments with immobilized αE-catenin on the glass slides, where the mBBr was introduced simultaneously with F-actin filaments. Cysteine labeling was achieved by a 20-minute incubation at 4 °C in the presence of 1.5 mM mBBr (ThermoFisher Scientific) in thoroughly degassed buffer containing 20 mM Hepes pH 7.5, 150 mM NaCl. For experiments without F-actin, 5 μM αE-catenin  was used. In the experiments with F-actin we employed αE-catenin at 2 μM with F-actin at 20 μM; this concentration insured saturation of αE-catenin given the estimated K−1 flow rates, with a 0.2% aqueous formic acid Phase A and 0.2% formic acid in acetonitrile for Phase B. The analytical column was an in-house hand packed C18 reverse phase column of ~15 cm in length using 2.4 μm particles with an I.D. of 100 μm. Peptide samples were ionized by a collision-induced dissociation method and further analyzed by an Orbitrap Elite mass spectrometer . Resulted RAW files contained LC-MS/MS peptide fragmentation data. Further analysis was accomplished by Byonic version 2.14.27 (Protein Metrics) of a search algorithm that used a targeted Fasta database for αE-catenin and allowed detection of the mBBr modification of cysteines. All data were filtered and presented at a 1% false discovery rate as described previously50. Additional post processing to determine the labeling efficiency was completed using custom scripts in MatLab to align peptides and to determine the ratio of spectral counts observed with and without the mBBr modification. To quantify the labeling ratio of the cysteines in αE-catenin, we used a total spectral counts (SC) approach51. In the SC method, the labeling efficiency for a given cysteine was determined by a ratio of the total number of mBBr-modified peptides over the total (mBBr-modified and non-modified) number of peptides containing this cysteine. We note that no labeling of lysine residues, which can react slowly with mBBr, was detected.Samples of αE-catenin excised from SDS gels were digested to peptides using trypsin/LysC . No reduction or alkylation of cysteines was performed. The resulting peptides were analyzed using Liquid Chromatography Mass Spectrometry (LC-MS). In LCMS experiments, peptides were injected onto a Nanoacquity Ultra Performance Liquid Chromatography (UPLC)  column and eluted at 450 nl minThe labeling efficiency or labeling ratio for each cysteine was calculated as a weighted mean from a series of independent experiments. The labeling efficiency from each replicate contributed to the weighted mean in proportion to the total number of the detected peptides that the given replicate contributed to the total number of peptides across a whole series. Statistical significance was determined by Welch’s T-test utilizing the R software package. We calculated the weighted standard error of the labeling ratio for each of the 12 cysteines. Furthermore, in SC analysis, the measurement error of the labeling efficiency for each cysteine was a function of the total number of detected peptides and was calculated as a signal-to-noise ratio (√n/n). The total error was calculated as a square average of the measurement and standard error.27 (in ref.22 the reducing agent was 1 mM TCEP rather than DTT). To avoid αE-catenin dimerization at higher concentrations, the monomer was placed in the cell at concentrations varying between 8–11 μM. The binding partner was injected at concentrations varying between 70–155 μM. Two 2 μl injections were followed by 30–34 9 μl injections. Experiments were performed at 25 °C. Data were analyzed with the Microcal Origin software. For baseline correction the heat signal at saturation was averaged and subtracted from all data points. For multiple measurements the weighted average was calculated.ITC experiments were performed in a VP-ITC calorimeter  in 20 mM HEPES pH 8.0, 150 mM NaCl and 1 mM DTT, the same buffer used in previous studies of αE-catenin binding to β-catenin and vinculin2+-nitroloacetic acid (NTA) functionalized slides, NHS-activated 45 × 75 mm glass slides  were first immersed in a solution of 0.01 M N-(5-amino-1-carboxylpentyl) iminodiacetic acid (NH2-NTA) in dimethylsulfoxide solution containing 0.07 M triethylamine for 20 hours at room temperature with stirring. The slides were then washed three times with dimethyl sulfoxide and twice with ethanol, with stirring, to remove excess unreacted reagents. The NTA-coated surfaces were then dried under a gentle stream of nitrogen and dipped in 50 mM NaOH for 5 minutes, and thoroughly rinsed with ultrapure water. The slides were dipped in 1 M NiCl2 aqueous solution for 1 hour, then rinsed with water and 1 mM ammonium citrate buffer pH 3.0 and finally kept in water until they were used.To prepare the Ni2+-NTA glass surface was passivated with 10% pluronic F-127  and 1% Bovine serum albumin (BSA)  for 1 minute and 5 minutes, respectively, with a phosphate-buffered saline (PBS) wash after each of these steps. Monomeric αE-catenin bearing an N-terminal 8-His tag at 5 μM concentration was then pipetted onto a circular area defined by a 2 cm diameter silicone O-ring  and incubated for 1-hour at 4 °C. Three washes of the glass slide with 10% Tween-20 in PBS were performed to remove unbound αE-catenin. The slides were then washed once with PBS and then stored overnight at 4 °C in F-buffer . Prior to rheometry experiments, the slides were washed again in fresh F-buffer.Prior to immobilization of αE-catenin, the Ni38 was used to create uniform shear on the immobilized αE-catenin. Simultaneous cysteine labeling of unsheared and sheared αE-catenin was performed by rotation of the rheometer cone at 0 s−1 or 6,000 s−1 shear rates, respectively, in the presence of 1.5 mM mBBr, 20 μM F-actin, and 50% glycerol in F-actin buffer for 20 minutes at 16 °C. Each experiment used 10 slides in order to obtain sufficient protein for subsequent analysis. The reaction was stopped by adding 5 mM reduced glutathione in PBS and incubating for 1 hour at 4 °C. The protein was then eluted from the glass slides with 5% SDS and 10 mM EDTA, combined, and subjected to SDS-polyacrylamide gel electrophoresis. Extracted αE-catenin (in the form of gel bands) was submitted for further mass spectrometric analysis at the Stanford Mass Spectrometry Facility.A rheometer with cone-plate geometry38 . The shear rate 39. The viscosity of an aqueous solution rises exponentially with increasing glycerol concentration, so glycerol was used to produce physiologically relevant forces.In a rheometer with a cone-plate geometry, a fluid held between a moving cone of a small angle and a flat surface plate experiences a uniform shear across the radius of the cone38 Fig. . The she−2 and the shear rate was 6000 s−1, which gave a shear stress of 120 Pa.A glass slide bearing immobilized αE-catenin in a 2 cm diameter circle was affixed to the stationary plate of a cone-plate DHR-3 hybrid rheometer , so that the glass slide surface became the surface of the rheometer plate. We introduced the labeling solution , which was held by surface tension within the circle. The labeling solution is not viscoelastic and behaves as a Newtonian fluid, and every protein molecule immobilized on the surface experiences the same shear. The measured viscosity of the labeling solution was 0.02 N s m35, corresponding to an area too small to experience significant forces under the conditions of our experiments. However, attachment of an actin filament will greatly increase the area of the αE-catenin•F-actin complex, and thus the forces imparted by fluid shear. Actin rods attached at a single end are the most likely to have a finite tilt angle relative to the surface, which would complicate calculation of the area subjected to shear. Attachment of an αE-catenin molecule elsewhere in the rod would lead to a smaller angle between the rod and the surface, and if a single rod is attached to multiple complexes it should lie flat on the surface. To assess the effect of the extreme case of an end-on attachment of a rod with a “free hinge”, we calculated the Weissenberg number of the actin rods 52, using the observation that purified G actin polymerized in F-buffer with phalloidin yields actin filaments with an average length of 4.9 μm53, and that the width of an actin filament is approximately 8 nm. With a shear rate of 6,000 s−1, the Weissenberg number is 6.5 × 105. Previous measurements on tethered DNA chains subjected to shear flow revealed tilt angles of less than a degree for Weissenberg numbers several orders of magnitude lower than were generated in the present experiments54. Thus, it is expected that the actin rods attached to αE-catenin in the presence of the shear rates employed here will essentially lie flat on the surface. We calculate the area of a filament as a rectangle, and the dimensions above give an area of 0.039 μm2. Using this area and the shear stress of 120 Pa, the force calculated from F = τ x A is 4.7 pN.To calculate the forces experienced by the complex, we first note that αE-catenin alone has a solution radius of 4.4 nm26, residues 273–635, was used in the simulations. The simulations were performed as previously described34, except that the equilibrium simulations were extended to 200 ns and the steered MD (SMD) simulation56 was performed for 2.2 μs in the presence of external force (100 pN).The M domain model from chain A of the published αE-catenin structure (PDB ID 4IGG)Supplementary Material


Forty-eight rats were randomly allocated to four groups (n=12/group): control, DNP, DEX, and yohimbine groups. The DNP model was established by streptozotocin (STZ) injection. The effects of DEX with or without the α2 adrenergic antagonist yohimbine were assessed by behavior tests . Spinal cord tissue was evaluated by immunofluorescence staining of astrocytes as well as for Wnt 10a and β-catenin expression, western blot analysis of Wnt 10a and β-catenin expression, and enzyme-linked immunosorbent assay measurement of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β). Rats with STZ-induced DNP had a decreased pain threshold, activated astrocytes, increased expression of Wnt 10a and β-catenin, and increased levels of proinflammatory cytokines compared to the control group, and these effects were ameliorated by treatment with DEX. Yohimbine administration partly abolished the protective effects of DEX in the DNP model rats. In conclusion, DEX alleviated DNP in rats by inhibiting inflammation and astrocyte activation, which may be attributed to downregulation of the Wnt 10a/β-catenin signaling pathway.Diabetic neuropathy pain (DNP), a spontaneous pain with hyperalgesia and allodynia, greatly compromises patients' quality of life. Our previous study suggested that dexmedetomidine (DEX) can relieve hyperalgesia in rats by inhibiting inflammation and apoptosis at the level of the spinal cord. In the present study, we aimed to evaluate the role of Wnt 10a/ Its chacurrence . Howeverα2 adrenergic agonist, exerts anti-inflammatory effects by regulating the balance between the sympathetic [Dexmedetomidine (DEX), a highly selective pathetic  and parapathetic . In our pathetic . Howeverβ-catenin signaling in the effects of DEX in a rat model of DNP. By revealing the specific mechanisms by which DEX inhibits glial activation and the inflammatory response, the results of this study can support further mechanistic studies as well as clinical applications of DEX in the treatment of DNP.The present study aimed to assess the role of Wnt 10a/Male Sprague–Dawley rats  were obtained from Guangdong Medical Laboratory Animal Center . The study protocol was approved by Guangzhou Medical University's Animal Ethics Committee.α2-adrenoceptor antagonist that blocks the active site of DEX.Rats were individually housed at a room temperature of 23–25°C without limit of access to food and water. A total of 48 rats were randomly allocated to four groups (n=12 in each group): a control group, a DNP model group (DNP group), a DEX-treated group (DEX group), and a yohimbine-treated group (YOH group). Yohimbine is a selective μg/kg DEX  [The rat DNP model was established as previously described . For all, China)  was injeThe behavioral tests included the MWT test for the mechanical stimuli response and the thermal withdrawal latency (TWL) test for the thermal stimuli response on days 29–45 after STZ injection.For the WMT test, we placed the rats in a Plexiglas chamber and used the von Frey filaments  to stimulate rats' plantar surface of the left hind paw. Each stimulation lasted for 3–5 s. A quick hind paw withdrawal was defined as a positive response. A force of 0.6 g was applied at the beginning, followed by ascending or descending consecutive stimuli; the force was reduced after a positive response and increased after a negative response. The minimal threshold force to induce the withdrawal response was finally determined. Notably, the force should not exceed 15 g to avoid tissue damage. We calculated the average MWT values from the results of 10 tests to get the 50% withdrawal threshold.For the TWL test, the thermal stimuli response of the left hind paw was measured using a full-automatic plantar analgesia system . Rats were placed on a 3-mm-thick glass plate within a Plexiglas chamber. Then, the left hind paw was exposed to a heat stimulus directly from a fixed distance. The elapsed time for withdrawal of the paw from the heat was recorded. The average value of five tests performed at 5-min intervals was calculated. A maximum cut-off value of 30 s was used to prevent possible damage.On days 39, 42, and 45, 350 mg/kg of 10% chloral hydrate was injected intraperitoneally and 250 mL normal saline was used for heart perfusion in rats (n=4 in each group). The spinal cord from L3–5 was isolated and stored.β-catenin  with a specific buffer  at 4°C overnight. After that, a horseradish peroxidase-conjugated goat/anti-rabbit secondary antibody  was used applied for incubation for 2 h at room temperature. GAPDH  was set as a loading control. The expression bands of target proteins were analyzed with the Quantity One software , and the densitometric values were used for analysis. Normalized protein expression levels were calculated relative to GAPDH expression levels.Tissues were homogenized, and proteins were extracted in lysis buffer  on ice. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), proteins were transferred to a polyvinylidene fluoride membrane. After blocking for 4 h, membranes were incubated with primary antibodies including a rabbit/anti-rat Wnt 10a  and a rabbit/anti-rat β-catenin  were applied for incubation at 4°C overnight. Then, sections were incubated with a biotinylated donkey/anti-mouse or anti-rabbit IgG  in 1.5% donkey serum  for 20 min at 37°C. Finally, a mixture of Alexa Fluor 532- and 488-conjugated secondary antibodies was added for incubation.Spinal cord segments were fixed and cut into frozen sections. Primary antibodies including a mouse/anti-rat primary antibody against glial fibrillary acidic protein (GFAP) , a rabbit/anti-rat Wnt 10a , and a rabbit/anti-rat/β-catenin was acquired from eight randomly selected sections and calculated with the ImageJ software . The quantification analysis was carried out by an independent researcher unaware of the treatments.A mixture of 50% glycerin in phosphate-buffered saline (PBS) was used to treat the sections. Images were recorded using a confocal laser scanning microscope . The mean fluorescence intensity for GFAP, Wnt 10a, and α and interleukin (IL)-1. The tissues were prepared using a grinder and an ultrasonic tissue homogenizer. The tissue supernatant was collected from centrifuged samples for analysis. The optical density (OD) values were assessed with a microplate reader  at 490 nm. The TNF-α and IL-1 levels were determined according to the kit directions.Enzyme-linked immunosorbent assay (ELISA) kits  were used to measure the proinflammatory factor levels including the tumor necrosis factor (TNF)- P<0.05.All statistical analyses were performed with SPSS software . Data are presented as the mean ± standard deviation (SD) and were compared with one-way analysis of variance, followed by the least significant difference test. Statistical significance was set at aAs shown in α2-adrenoceptor /transcriptional coactivator with the PDZ-binding motif (TAZ), integrates into β-catenin and regulates the Wnt pathway response. Studies have shown that blocking or knocking down YAP/TAZ expression in the spinal cord reduces neuronal damage and Wnt-induced mechanical hyperalgesia [β-catenin signaling in the astrocytes and elevating inflammatory cytokine levels [β-catenin downstream. A recent study found that DEX inhibited the nuclear translocation and binding activity of activated NF-κB, thus reducing inflammatory cytokines [β-catenin expression was suppressed, and the secretion of inflammatory cytokines was reduced.Activation of the classical Wnt/ralgesia . In addiralgesia . In the e levels . A recene levels . Consistytokines . Our resGlial cells are the largest group of cells in the nervous system, and they include astrocytes, microgliacytes, NG2 (nerve/glia antigen 2) cells, and oligodendrocytes. In the central nervous system, astrocytes provide neurotrophic support and are important regulators of neurotransmitter functions. Astrocytes, together with microglia, play an important role in neuroinflammation and various neuropathophysiological signal processes. On one hand, intercellular substance exchange happens between the glial cells through functional syncytia formed by gap junctions and neurons. On the other hand, neuronal activity and synaptic plasticity are regulated by the intracellular calcium concentration, the adenylate cyclase-cAMP delivery system, and a remote cell–cell signaling . In normβ-catenin signaling. Thus, the use of DEX may block the positive feedback regulation between the glial cells and inflammatory responses.In the spinal dorsal horn, astrocytes and microglia are activated upon many forms of stimulation including infection, inflammatory mediators, ischemia, apoptosis, and mechanical compression or injury. These activated cells in turn release large amounts of inflammatory cytokines, nerves growth/trophic factors, and neurotransmitters . The actβ-catenin signaling in association with DEX treatment. The underlying mechanisms need to be investigated in the future.In our previous study, hyperalgesia was noted in DNP rats with the activation of microglia but not astrocytes. However, astrocytes were significantly activated in the present study. This difference is due to the different time points for observation. Microglia is significantly activated in the initial stage of chronic pain, followed by the activation of astrocytes . The actβ-catenin signaling pathway.In summary, DEX reduced DNP in rats by inhibiting inflammation and astrocyte activation, possibly through the Wnt 10a/


An effective approach to the fabrication of progressive epoxy nanocomposites by the incorporation of hydroxyl-terminated dendrimers functionalized graphene oxide (GO-TCT-Tris) is reported. The relationship between surface grafting, chemical construction, morphology, dispersion, and interfacial interaction as well as the corresponding mechanical properties of the composites were studied in detail. It was shown that hydroxyl-terminated triazine derivatives have been resoundingly bonded onto the GO surface through covalent bonding, which effectively improved the dispersion and compatibility of GO sheets in epoxy resin. The tensile and flexural tests manifested that the GO-TCT-Tris/epoxy composites exhibited greater tensile/flexural strength and modulus than either the pure epoxy or the GO/epoxy composites. For GO-TCT-Tris (0.10 wt%)/epoxy composite, the tensile strength and elastic modulus increased from 63 ± 4 to 89 ± 6 MPa (41.27%) and from 2.8 ± 0.1 to 3.6 ± 0.2 GPa (28.57%), and the flexural strength and modulus increased from 106 ± 5 to 158 ± 6 MPa (49.06%) and from 3.0 ± 0.1 to 3.5 ± 0.2 GPa (16.67%), respectively, compared to the pure epoxy matrix. Moreover, the fractographic analysis also illustrated the ameliorative interfacial interaction between GO-TCT-Tris and epoxy matrix. Epoxy resins are typical thermoset plastic with eminent mechanical properties, chemical stability, corrosion resistance, and insulation behavior that can be extensively used in various applications, such as adhesives, coatings, electronic packaging, and the automotive and the aerospace industry ,2. HowevGraphene and graphene oxide (GO), a novel two-dimensional nanomaterial, has given rise to considerable theoretical and technological researches on polymer composites due to its fantastic and unparalleled physical properties, including a high specific surface area, high fracture strength, elastic modulus, and thermal conductivity ,9. It haIt has been reported in our recent publications that functionalization of GO to generate derivatives could improve their dispersion and compatibility properties in the resin matrix ,16,17. CThese approaches have demonstrated good results, but some deficiencies still existed, such as high cost, high energy consumption, low efficiency, and even GO or graphene structure destruction . In contLow-cost tris(hydroxymethyl)aminomethane (Tris) has distinct polar features with abundant hydroxyl and amine groups, which could react with the active site on the basal planes and edges of GO and could even participate in a curing reaction with epoxy resin , which w3), potassium permanganate (KMnO4), hydrogen peroxide (H2O2), cyanuric chloride (TCT), tris(hydroxymethyl)aminomethane (Tris), triethylamine (TEAE), lithium aluminum hydride (LiAlH4), and tetrahydrofuran (THF) were acquired from Tanzhi Co. Ltd. . Concentrated sulfuric acid (98% H2SO4) and hydrochloric acid (37% HCl) were bought from Sinopharm Chemical Reagent Co., Ltd.  and were analytical grade.Graphite powder was offered by Qingdao Tianhe Co. Ltd.  Epoxy resin  was used as the matrix in the composite. Liquid aromatic amine type epoxy resin hardener 3,3-diethyl-4,4-diamino diphenyl methane (H-256) was supplied by Shanghai Macklin Biochemical Co.  Sodium nitrate . The GO–OH was dispersed in THF with sonication for 1 h. Under stirring, the mixture was added to TCT and TEAE to absorb the acid produced in the reaction. After stirring at 70 °C for 24 h, the mixture was rinsed thrice by THF to remove the TEAE and ethanol. The suspension was dried under vacuum conditions to get functionalized GO (GO-TCT). The GO-TCT was dissolved in 60 mL of acetonitrile, then Tris and TEAE were added. The mixture was heated and refluxed at 80 °C with stirring for 12 h. Finally, it was rinsed with ethanol and dried to get Tris-functionalized GO-TCT (denoted as GO-TCT-Tris). The preparation route of GO-TCT-Tris is shown in The prepared GO was firstly dispersed in LiAlHThe fabrication process of GO-TCT-Tris/epoxy composites is presented in −1 and wavenumber at the 4 cm−1 resolution. X-ray photoelectron spectroscopy  was used to characterize the surface chemical composition and elemental content of the sample. The chemical composition of the sample was investigated by X-ray diffraction . The scanning range was from 5° to 45° with a scan rate of 3°·min−1. Raman spectra were analyzed using a 633-nm laser and recorded using a LabRAM spectrometer . TGA-DTG analysis was carried out by STAR SW in the temperature region of 25 to 800 °C and heating rate of 5 °C/min in a nitrogen atmosphere. Scanning electron microscopy  was carried out to investigate the surface morphology of samples. The dispersity of GO in the matrix was observed by transmission electron microscopy  and optical microscopy. The tensile and flexural tests were examined on a universal testing machine  with a crosshead speed of 2 mm min−1 and gauge length of 60 mm. In addition, the specimen dimensions were 80 mm (long) × 10 mm (wide) × 4 mm (thick). Each specimen was tested 5 times to calculate the average value.Fourier transform infrared spectroscopy  was conducted to analyze the functional groups on the surface of the graphene and hybrid materials, which were recorded in the wavelength range of 500 to 4000 cm−1, C=O from carboxyl at 1716 cm−1, C=C from the aromatic ring at 1682 cm−1, and C–O–C from epoxy at 1038 cm-1 in -1 corresponding to the C–N bond of the triazine ring and the C–Cl stretching vibrations in The surface groups on the surface of functionalized GO sheets were measured by FTIR, as shown in −1) and G band (1585 cm−1), as shown in 2 bond structure [D/IG is a measured way of disordered graphite. The ID/IG ratio of GO was 1.74, manifesting that there were some structural defects. The ID/IG of GO-TCT dropped down to 1.63 because the TCT had a stable six-membered heterocyclic ring structure that filled up the 2sp area of GO. After Tris modification, the ID/IG value of GO-TCT-Tris increased slightly to 2.34, which was because some chemical bonds and hydrogen bonds were formed on the GO surface [2 structure of graphene did not show severe damage during the modification process.The internal structure of the different sheets was characterized by Raman spectroscopy. The Raman diffraction spectrum of GO and its derivatives mainly includes two bands, D band , C-C (~284.7 eV), C–OH (~285.6 eV), C–O–C (~286.8 eV), and O–C=O (~288.6 eV) in N1s spectra were used to further illustrate the chemical reaction of Tris between the GO-TCT surfaces, as exhibited in The exfoliation level of GO, GO-TCT, and GO-TCT-Tris was characterized by XRD, as shown in D/IG values and layer spacing as mentioned in Raman and XRD.The structural morphology and surface characteristics of GO derivate were observed through SEM and TEM, as displayed in The TEM image of GO derivate sheets presented a natty and transparent surface with a few thin ripples, as shown in 4, manifesting that the TCT molecules grafted on the GO surface, preventing decomposition of the carbon skeleton of GO. Comparatively, for GO-TCT-Tris, at 800 °C, the total mass loss was about 40%; Based on the TGA result, the mass fraction of Tris molecules grafted on the GO sheets could be acquired by calculation, which was ~7%.To ascertain the thermal stability of GO, GO-TCT, and GO-TCT-Tris as well as the relevant information of the TCT and Tris grafted to the GO sheets , TGA andIt is well known that nanofillers (such as GO and carbon nanotube) with a high specific surface area are inclined to attract each other to form clusters even within the uncured polymer owing to the van der Waals forces and Coulomb attractions . The disRepresentative stress–strain curves for neat epoxy and its nanocomposites, along with the results of tensile and flexural testing, including tensile strength, elastic modulus, elongation at break, flexural strength, and modulus, are listed in The interface quality improvement of the GO-TCT-Tris/epoxy composites could be attributed to two aspects: (1) Strong covalent bonding between hydroxyl-terminated triazine derivatives and the GO sheet resulted in the quantity of linked peripheral hydroxyl groups increasing in the presence of hydrogen bonds, which could further react with the epoxy resin, thus forming chemical bonding in the interface to increase the interfacial adhesion and compatibility with the epoxy matrix. Moreover, the hyperbranched triazine derivatives not only enlarged the specific surface area but also had more interspace in its inner and outer, which were penetrated by the epoxy matrix to generate strong mechanical interlocking. To further estimate the dispersion and interface of GO-TCT-Tris/epoxy composite nanocomposites, the cross-sectional fracture surfaces of the specimens after tensile testing were observed by SEM. For 0.10 wt% GO/epoxy composites, there were many ripples structures on the fracture surface a, which In summary, GO functionalization with low-cost hydroxyl-terminated dendrimer was testified to improve the dispersion/exfoliation and compatibility in epoxy resin. The tensile and flexural strength of GO-TCT-Tris/epoxy composites was significantly enhanced (increased by 41.27% and 49.06%) compared with that of the neat epoxy. The fracture surface morphologies demonstrated that the hydroxyl-terminated dendrimers’ functionalization contributed to better dispersed/exfoliated sheets and stronger interfacial interaction between GO sheets and the epoxy matrix, which facilitated effective stress transfer from the matrix to the GO-TCT-Tris sheets.


Objective: The Wnt/β-catenin pathway is involved in the development of hepatocellular carcinoma (HCC) and malignant events such as the epithelial-mesenchymal transition (EMT), metastasis, and invasion. Studies have illustrated that the inhibition of tankyrases (TNKS) antagonizes Wnt/β-catenin signaling in many cancer cells.Methods: The expression levels of proteins related to the Wnt/β-catenin pathway and EMT were analyzed by immunohistochemistry in HCC tissue and paired adjacent normal tissue (n = 10), and in an analysis of The Cancer Genome Atlas (TCGA) data. Additionally, after treatment of HCC cell lines with TNKS1/2 small interfering RNA (siRNA) and a novel TNKS inhibitor (NVP-TNKS656), cell viability, cell clone formation, wound-healing, and cell invasion assays were performed.Results: Higher expression of β-catenin, TNKS, vimentin, and N-cadherin was observed in HCC tissue compared to adjacent normal tissue, but lower expression of E-cadherin was found in HCC tissue. These findings were also observed in the TCGA analysis. In addition, TNKS inhibition (using TNKS1/2 siRNA and NVP-TNKS656) not only abrogated the proliferation of the HCC cell lines but also suppressed metastasis, invasion, and EMT phenotypic features. Moreover, the mechanisms related to TNKS inhibition in HCC probably involved the stabilization of AXIN levels and the downregulation of β-catenin, which mediates EMT marker expression.Conclusion: The TNKS/β-catenin signaling pathway is a potential anti-proliferation and anti-metastatic target in HCC. Hepatocellular carcinoma (HCC), which has poor prognosis and a high mortality rate, is one of the most common causes of cancer-related death in the world Two tankyrase (TNKS) isoforms,TNKS1 and TNKS2, belonging to a group of enzymes called poly ADP ribosyl polymerases (PARPs) With the advent of novel inhibitors of TNKS, TNKS can act as a novel target in various cancers. The TNKS inhibitors XAV939 and WXL-8 attenuate WNT/β-catenin signaling and inhibit HCC cell growth TNKS, β-catenin, AXIN, vimentin, E-cadherin, and N-cadherin antibodies were purchased from Sigma-Aldrich and Abcam . NVP-TNKS656 was purchased from CSNpharm .2.The HCC cell lines SMMC-7721 and MHHC-97h were purchased from ATCC and cultured in Dulbecco's Modified Eagle Medium  containing 10% heat-inactivated fetal bovine serum  and 2 mM L-glutamine (Gibco). Both cell lines were maintained in an incubator at 37°C in a fully humidified atmosphere of 5% COTen HCC samples with adjacent tissue samples were obtained from 10 HCC patients at the Second Xiangya Hospital, Central South University. Informed consent was obtained and the study was approved by Ethics Committee of the Second Xiangya Hospital (no. 2019026-18).http://ualcan.path.uab.edu) and TCGA HCC subgroup data of individual stage. Heat maps of differentially expressed genes in HCC and adjacent normal tissues were created. Each gene expression level was represented as log2 (transcript count per million [TPM]+1). Box-whisker plots were used to show the gene expression in the HCC subgroup compared to adjacent normal tissues. Gene-level correlations with patient overall survival were also conducted. The TCGA HCC patient survival data were used for Kaplan-Meier survival analyses and for generating overall survival plots in the UALCAN web-portal.TNKS/β-catenin pathway-related genes and EMT-related genes  were analyzed using the UALCAN web-portal  and PDB (http://www.rcsb.org/), respectively. The docking analysis was conducted as described in the previous study The structure of the TNKS inhibitor NVP-TNKS656 (CID 136237316) and TNKS1 (Protein Data Bank [PDB] code 4LI6) were obtained from PUBCHEM  alone (used as the vehicle control) or various concentrations of NVP-TNKS656 (from 1.25 to 100 μM). After 3 days of incubation, the media and compounds were removed and replaced with 100 μl fresh DMEM and 20 μl Cell Counting Kit (CCK)-8 Reagent . After incubation for 2 h, the absorbance at 450 nm was measured using a microplate spectrophotometer.Pairs of independent double-stranded TNKS1 and TNKS2 siRNAs were identified in a previous study and synthesized by GenePharma Co. Ltd. . The siRNA sequences were as follows: TNKS1 sense strand 5'-GCAUGGAGCUUGUGUUAAUUU-3' and anti-sense strand 5'-AUUAACACAAGCUCCAUGCUU-3'; TNKS2 sense strand 5'-GAGGGUAUCUCAUUAGGUAUU-3' and anti-sense strand 5'-UACCUAAUGAGAUACCCUCUU-3'. To prepare the siTNKS1/2 mixture, the TNKS1 and TNKS2 siRNA were denatured at 95 °C for 5 min, annealed at room temperature for 30 min, and then mixed together to create the siTNKS1/2 mixture. Transient transfections of 50 nM siTNKS1/2 mixture and scrambled siRNA (siRNA negative control [siNC]) were accomplished using Lipofectamine 2000 reagent  according to the manufacturer's protocol.HCC cell lines treated with NVP-TNKS656 or transfected with the siTNKS1/2 mixture were reseeded in a new six-well culture plate at a density of 200 cells/well and incubated for 7-9 days. Fresh DMEM containing various concentrations of NVP-TNKS656 (0.125-20 μM) was added every 2 days. The six-well plate was then washed with phosphate-buffered saline (PBS) and colonies were stained with 0.5% crystal violet in PBS.For the assessment of the migration ability of HCC cells, SMMC-7721 and MHHC-97h cells were seeded in six-well plates with DMEM containing 5% FBS and grown to 90% confluence. After the media were aspirated, a yellow pipette tip was used to make a straight scratch in each monolayer. Subsequently, the cellular debris was washed with PBS twice. The cells were exposed to NVP-TNKS656 or transfected with siTNKS1/2 for 24 h, respectively, as described above.5 cells/ml) were added to the matrigel-coated upper chamber and incubated for 36 h. After aspirating the medium from the upper chamber and washing twice with PBS, the cells were permeabilized with 100% methanol for 20 min at room temperature. The methanol was then aspirated and the chambers were washed twice with PBS. Non-invaded cells were scraped off with cotton swabs. The invasive cells were stained with 0.5% crystal violet for 20 min. After the crystal violet was removed and the invasive cells were washed twice with PBS, the invasive cells in four random fields under a bright light microscope were counted.One hundred microliters of matrigel (0.4mg/ml)  was loaded into the upper chamber of a Transwell plate  and incubated for 24 h. HCC cell lines were treated with NVP-TNKS656 or siTNKS, as described above, and then trypsinized to create a cell suspension. For the lower chamber, 750 µl DMEM with 10% FBS was added. Meanwhile, 200 µl cell suspension cells  buffer (Beyotime). Protein concentrations were assayed using bicinchoninic acid (BCA) protein assay reagent (Beyotime). The western blotting process was based on a previous report SPSS 13.0 statistical software  was used for the statistical analyses. Quantitative variables were expressed as mean ± SD and analyzed with t tests. P-value <0.05 was considered statistically significant.p=0.0011). Kaplan-Meier plots showed that only β-catenin expression was associated with HCC patient overall survival (p<0.05). As shown in Figure As shown in the heatmap and box-whisker plots of genes in Figure The TNKS inhibitor NVP-TNKS656 and TNKS knockdown using siRNA were used to investigate the effect of TNKS/β-catenin signaling on the proliferation of HCC cell lines. As shown in Figure Given the essential role of TNKS regarding regulating the Wnt/β-catenin signaling pathway in HCC cells, TNKS1/2 knockdown was investigated to assess whether TNKS plays a positive regulatory role in the metastasis, invasion, and EMT characteristics of SMMC-7721 and MHCC-97h cells. As shown in Figure Similar results were also found for the cell invasion assay. The cell invasion assay showed that TNKS1/2 knockdown by siRNA transfection significantly suppressed the cell invasion ability compared to cells transfected with scrambled siRNA (siNC). Further, the western blotting results showed that TNKS/β-catenin signaling was clearly inhibited by TNKS1/2 siRNA transfection in MHCC-97h cells, as indicated by the downregulation of β-catenin and TNKS1/2, as well as the upregulation of AXIN1/2. The EMT genes, N-cadherin and vimentin, were also decreased after TNKS1/2 siRNA transfection or combination with 2.5 μM NVP-TNKS656, along with elevated levels of E-cadherin. These findings indicate that TNKS1/2 knockdown was responsible for β-catenin downregulation and the further abrogation of metastasis, invasion, and EMT in HCC cell lines.To further explore the use of TNKS as a therapeutic target for the inhibition of metastasis, invasion, and EMT in HCC cell lines, NVP-TNKS656, a TNKS inhibitor, was used to treat HCC cell lines. As shown in Figure The present study showed that the aberrant expression of TNKS/β-catenin pathway-related genes and EMT markers in HCC patients could act as therapeutic targets. TNKS inhibition in HCC cell lines (by siRNA or the inhibitor NVP-TNKS656) further indicated that the TNKS/β-catenin signaling pathway is an anti-proliferation and anti-metastatic target in HCC cell lines.Numerous studies have shown that the canonical Wnt/β-catenin signaling pathway plays a vital role in HCC EMT is an essential step in HCC progression that potentiates malignant tumor cell invasion and metastasis, thereby leading to death in HCC patients TNKS1 and TNKS2, positive regulators of β-catenin activation, were reported to be overexpressed in HCC samples Regarding to the roles of TNKS1 and TNKS2 as positive regulators of β-catenin pathway activation, many TNKS inhibitors have been discovered and characterized Taken together, our results suggest that TNKS inhibition decreased the invasion and metastasis capacity in HCC cell lines by affecting β-catenin-mediated EMT. The related signaling pathway may offer potential targets for HCC cancer therapy. However, β‑catenin inhibitors may also be applicable for the prevention of organ fibrosis, second‑line HCC prevention, and treating β‑catenin‑driven cancer. Multilayered prevention and treatment strategies for β‑catenin‑related human cancers are necessary for the implementation of personalized precision medicine.


IKKβ, an essential kinase of NF-κB signaling, is composed of an N-terminal kinase domain (KD) and a C-terminal scaffolding domain, containing a ubiquitin-like domain (ULD). The Hsp90 chaperon has special responsibility for folding of protein kinases including IKKβ. Here, we found that Hsp90 inhibition induced IKKβ degradation, which is partially mediated by Keap1. Geldanamycin (GA), a Hsp90 inhibitor, enhances association of IKKβ with Keap1 through the binding site in KD, and translocates IKKβ to detergent-insoluble fractions leading to its autophagic degradation. An electrophile tBHQ suppressed Keap1-mediated proteasomal Nrf2 degradation but not autophagic IKKβ degradation. Substitution mutation of Leu353 to Ala in the ULD destabilizes IKKβ, enhances its association with Keap1, translocates it to detergent-insoluble fractions, and causes its autophagic degradation. These results suggest that Keap1 is involved in the degradation of structural destabilized IKKβ and negative regulation of NF-κB under proteotoxic stress. NF-κB is a critical transcription factor, regulating oxidative stress response, inflammation and carcinogenesis , 2. In rMaintenance of proteome integrity and protein homeostasis depend on a complex network of molecular chaperones, including conserved heat shock proteins (HSPs) , 12. EnvKeap1 is a ubiquitin ligase which interacts with a transcription factor Nrf2, a master regulator of the antioxidant response , 21. UndInhibition of HSPs or heat shock leads to proteotoxic stress that causes cellular protein damage or conformational changes such as protein misfolding, resulting in protein degradation. IKKβ degradation caused by Hsp90 inhibition may reflect an important adaptive cellular response which links proteotoxic stress response to inflammatory stimuli. However, the mechanism of IKKβ degradation by Hsp90 inhibition is yet be studied. In this study, we investigated the mechanism by which Hsp90 inhibition causes IKKβ degradation and found that structural destabilization of IKKβ enhances its binding to Keap1 and promotes its autophagic degradation by different mechanism from that of Nrf2.Anti-tubulin (H-300) and anti-HA (Y-11) rabbit antibodies were obtained from Santa Cruz Biotechnology. Anti-HA (11867423) rat monoclonal antibody and anti-Keap1 (10503-2-AP) rabbit antibody were purchased from Roche and Proteintech, respectively. Anti-IKKβ (2684), anti-phospho-IKKβ (2697) and anti-phospho-IκBα (2859) rabbit antibodies were procured from Cell Signaling. Anti-Flag (M2) mouse antibody was obtained from Sigma. MG132, Chloroquine, GA, 3-methyladenine(3-MA), and cycloheximide (CHX) were procured from Sigma-Aldrich. Tertiary Butylhydroquinone (tBHQ) was purchased from Tokyokasei.Keap1-/- MEFs were prepared in the laboratory of Masayuki Yamamoto. Keap1 and Nrf2 were amplified from a human cDNA library by polymerase chain reaction (PCR), and cDNAs were cloned in expression vectors encoding HA and Flag sequences. Expression plasmids encoding IKKβ, IKKα, NEMO/IKKγ and IκBα were described previously [Human embryonic kidney (HEK293) and mouse embryonic fibroblast (MEF) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin. HEK293 cells were obtained from RIKEN cell bank. WT and eviously . Substit2, 60 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM 4-amidino phenyl methyl sulfonyl fluoride, 50 KIU/ml aprotinin, 20 μg/ml pepstatin, 20 μg/ml leupeptin, 2 mM dithiothreitol and 1% Triton X-100. After centrifugation at 16,000 x g for 20 min at 4°C, the lysates were fractionated into supernatants and pellets. The pellets were then solubilized in buffer B containing 100 mM Tris-Cl, pH 6.8, 2% SDS, 5% glycerol, and 2.5% 2-mercaptoethanol. Cells solubilized in buffer B were used as total cell lysates.Cells were solubilized in buffer A consisting of 20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 10 mM EGTA, 10 mM MgClFor the immunoprecipitation assay of transfected cells, cell supernatants were incubated with anti-Flag (M2) Sepharose (Sigma), or with an antibody combined with Protein A and Protein G Sepharose  at 4°C. For the immunoprecipitation assay of endogenous proteins, cell lysates were incubated with an antibody together with TrueBlot anti-mouse or anti-rabbit IP beads (eBioscience) and subjected to immunoblotting using TrueBlot HRP-conjugated anti-mouse or anti-rabbit IgG antibodies (eBioscience). Proteins separated by gel electrophoresis were transferred to polyvinylidene difluoride membranes (Millipore) by an electroblotting apparatus  and subjected to immunoblotting using HRP-conjugated anti-mouse or anti-rabbit IgG antibodies . Antigen-antibody complexes were detected using SuperSignal West Pico Chemiluminescence System (Pierce).6 cells) were transfected with 0.2 ug plasmids encoding HA-IKKβ, HA-IKKβ (L353A), Flag-Keap1, and GFP-LC3. Cells were grown on collagen-coated coverslips. After 18 h, cells were fixed with 4% paraformaldehyde in PBS for 30 min, and then fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature. After washing with PBS, cells were stained with anti-HA rat monoclonal antibodies or anti-Flag (M2) mouse antibodies in Can Get Signal immunostain Immunoreaction Enhancer Solution (TOYOBO) for 2 h. After washing with PBS, coverslips were incubated with Cy3-conjugated anti-mouse IgG antibodies or FITC-conjugated anti-rat IgG antibodies (Jackson ImmunoResearch Laboratories) for 2 h and were observed under BZ-9000 Fluorescence Microscope (KEYENCE).HEK293 cells (0.5 x 10Data were expressed as means   standard deviation (S.D.) from at least three independent experiments and analyzed by two-tailed t-tests. Differences were considered statistically significant if at * P < 0.05, ** P < 0.01.Keap1-/- MEF cells than in their wild-type counterparts, suggesting that Keap1 is partially involved in GA-induced IKKβ degradation  or Atg8-interacting motif (AIM) in the N-terminal region which recruits the IKK complex to the autophagosome, and acts as a selective autophagy receptor that mediates the degradation of the IKK complex . WhereasCells are challenged by various extrinsic and intrinsic insults which provoke proteotoxic stress under conditions of injury, inflammation, and oxidative stress. As disturbance of proteome homeostasis is associated with a broad range of human diseases, maintenance of proteome integrity is essential to maintain cellular fitness and functions. To protect cells from proteotoxic stress, cells mobilize heat shock proteins including Hsp90 that unfold proteins and proteolysis systems that remove damaged proteins. In this study, it was found that Hsp90 inhibition, which causes proteotoxic stress, enhances interaction of IKKβ with Keap1. These findings also suggest that cells respond to proteotoxic stress by regulating IKKβ/NF-κB and Keap1/Nrf2 signaling pathways. The Keap1/Nrf2 and IKKβ/NF-κB signaling pathways have interesting features concerning the proteolytic mechanisms such as autophagy and UPS. Keap1 suppresses Nrf2 activation by UPS, whereas it is degraded by autophagy in response to oxidative stress , 27. IKK


Caducity is known to be an independent risk factor in osteoarthritis (OA), yet the molecular basis behind caducity and OA remains unclear. Klotho, an anti-caducity protein, is an endogenous antagonist of the transduction of Wnt/β-catenin signal which can stimulate the articular cartilage degradation, indicating that deficiency in Klotho may increase Wnt/β-catenin activity and consequently accelerate the development of OA. We found that expression of Klotho was markedly higher in normal mouse cartilage than in the OA model, and in this model the activity of Wnt/β-catenin and its target gene was up-regulated. Decrease in Klotho expression was closely associated with the increase of β-catenin in OA, indicating that there was a negative correlation between Klotho and Wnt signal transduction. In the vitro and in vivo experiments, Klotho was found to bind to multiple Wnt, including Wnt1, Wnt4 and Wnt7a. It was additionally found that cyclic tenisle strain (CTS) inhibited the expression of Klotho and activated β-catenin. On the contrary, over-expression of Klotho would reduce the degradation of articular cartilage induced by CTS. These results suggest that Klotho is an antagonist of endogenous Wnt/β-catenin activity. In OA cartilage, decrease in expression of Klotho can activate Wnt/β-catenin signal transduction and consequently induce cartilage injury. Osteoarthritis (OA), characterized by sub-chondral synovial activation, bone remodeling, and progressive loss of cartilage homeostasis, is generally regarded as the primary age relevant osteoarticular disease , 2. The Klotho, named after a Greek goddess Klotho, was identified as a gene mutated in Klotho mice in 1997, which is primarily expressed in reproductive organs, kidney and brain , 6. MiceThe mechanism underlying the protection of cartilage from Klotho still remains unclear. Previous studies suggest that in renal injury model, Klotho expressed in renal tubular epithelial cells can bind to Wnt1, 4 and 7a to form a complex, which inhibits the activation of Frizzled and LRP5/6 complex, and plays a similar role to Wnt1, 4 and 7a antagonist. In this study, we hypothesized that Klotho would play a similar role in chondrocytes .In the pathogenesis of OA, the activation of classical Wnt signal transduction is observed in articular cartilage and injured synovium, and the expression of Wnt ligand and target gene increases –17. The Our experiment verified that low expression of Klotho in chondrocytes in OA also lost its antagonistic effect on Wnt1, 4 and 7a, which resulted in relative activation of Wnt/β-catenin and high expression of target gene MMP-2 and MMP-9. Finally, the degradation of the cartilage matrix was caused.The activation of Wnt/β-catenin signal transduction in articular chondrocytes leads to chondrocyte hypertrophy and ECM degradation. Klotho regulation of Wnt/β-catenin signal may improve the development of OA. This study aims to determine whether the reduction of Klotho expression in OA may induce the over-expression of Wnt/β-catenin signal and the degradation of cartilage matrix, and whether Klotho is a key factor in the pathogenesis of OA under this condition.Findings of representative photomicrographs HE staining and acin blue staining of joint pathological sections of normal mouse joints and OA model mice were available twelve weeks after ACLT. Intact and smooth cartilages were showed on sham-operated knee joints , 1B. AddTo further examine the expression of Klotho in OA, a mouse model was established. Cartilage tissue was removed from the knee joint with anterior cruciate ligament transection, and then the expression of Klotho in articular cartilage of OA mice was observed. The results showed that the level of Klotho in OA mice decreased markedly , 2D afteOn the contrary, the increased expression of Wnt signaling pathway and its target gene MMP2 and MMP9 in the cartilage of OA mice, and the increased expression of Phos-GSK3β (S9), Wnt1, Wnt4 and Wnt7a were observed by western blot, indicating that the Wnt signaling pathway was activated in OA .Wnt/β-catenin signal transduction pathway regulates the expression of MMPs gene downstream , 19. MMPThe β-catenin, the core protein in the Wnt signaling pathway, was transferred from the cytoplasm to the nucleus after activation. The results showed that in the OA model, when compared to the normal group, the β-catenin in the nucleus increased  and the Dishevelled (Dvl), a key protein in the Wnt signaling pathway, can transfer the Wnt signal from the receptor to the downstream effector. In the classical Wnt signaling pathway, Dvl is convoked to the cell membrane by the receptor Fizzled, and wherefore binds to the axin/GSK3/APC complex, which consequently inhibits the degradation of β-catenin in the cytoplasm. In the cartilage tissue of OA mouse model, the Dvl and Fizzled binding markedly increased  by Co-IPPrevious experiments suggest that Klotho can bind with Wnt1, 4 and 7a to form a complex, inhibits the activation of Frizzled, LRP 5/6 complex by Wnt, and therefore plays a similar role to Wnt1,4,7a antagonist. In the OA model, we also found that Klotho and Wnt1, 4 and 7a binding decreased when compared to normal cartilage tissue .ChIP method was used to detect the binding of β-catenin to MMP 2, 9 promoters. The results showed that the combination of TCF and MMP 2,9 promoters increased in the OA model , 3E.in vitro. Klotho protein level in the supernatant of chondrocytes was decreased with the increase of CTS intensity  in cells, and Wnt1, Wnt4 and Wnt7a) in the supernatant . Over-exIn the chondrocytes overexpressed with Klotho, Co-IP experiments confirmed that Klotho reversed the combination of Dvl and Frizzled induced by CTS . On the β-catenin is a multifunctional protein in the cytoplasm and a key molecule regulating gene transcription in Wnt signaling pathway. First, the chondrocyte was transfected with β-catenin low expressing lentiviral vector or negative control lentivirus vector. The results showed that there was no markable change in the expression of Klotho in the supernatant of chondrocytes between the β-catenin silencing chondrocytes and the normal cells, both decreased . HoweverIn the β-catenin silencing chondrocytes, Co-IP assay confirmed that there was no markable change in the binding of Dvl to Frizzled with or without CTS stimulation . SilenciOA is a common multivariate joint disease, in which caducity and overloading are main risk factors. Although the pathophysiology of OA involves cartilage, bone and synovial tissue, the primary feature of OA is the progressive degeneration of articular cartilage. Studies suggest that the progressive joint destruction in OA is caused by over-activation of Wnt signal transduction –17. The In many diseases, the down-regulation of Klotho increases the inflammation and oxidative stress –27. OA iin vitro and vitro experiments of OA cartilage tissue, Klotho can also be combined with Wnt1, 4, 7a , such as MMP-2 and MMP-9), are direct transcriptional targets of Wnt/β-catenin signaling pathway , 18. MMPIn further study, the binding of Wnt1, 4 and 7a to Klotho in chondrocytes increased significantly in cell supernatant and cells after chondrocyte over-expression of Klotho by using Klotho over-expression lentivirus vector. Under the action of CTS, Klotho reversed the activation of WNT/β-catenin signaling pathway , 7, indiin vivo and in vitro, the content of β-catenin in the nucleus increased and over-expression of Klotho could reduce the β-catenin transfer into the nucleus. Further study showed that after the β-catenin in chondrocytes was silenced by low expression of lentivirus vector, the reduced Klotho could not increase MMP expression through Wnt signaling pathway under the action of CTS , GSK3β, MMP2, MMP9, Wnt1, Wnt4, Wnt7, Frizzled and β-catenin were provided by Abcam . Anti-Dvl Antibody was provided by Cell Signaling Technology . Antibodies against Klotho was provided by Abcam and Santa Cruz Biotechnology Fetal bovine serum and DMEM low- and high-glucose medium were procured form HyClone  Phosphate-buffered saline (PBS), cytoplasmic and membrane protein extraction kit, total protein extraction kit, RIPA buffer and PMSF were procured from Beyotime Biotechnology . ChIP Assay Kit was obtained from Beyotime Biotechnology, . Pierce™ Classic Magnetic IP/Co-IP kit was obtained from Thermo Fisher . Acin blue staining kit was provided by Nanjing Jiancheng Bioengineering Institute .Twenty-four C57/6J mice  were obtained from the Animal Inc., affiliated to Nanjing Medical University . These mice were separated into two groups randomly (control group and OA group respectively), consisting of 12 mice for each, which were kept in four animal cages (six mice per cage) in a temperature-controlled room (21–23°C) with free access to water and food. All these animal procedures were approved by the Animal Research Ethics Committee of Nanjing Medical University.ACLT surgery was used to induce OA in adult male C57/6J mice. Briefly, all mice were anesthetized with chloral hydrate. A medial parapatellar approach was adopted to expose the right knee joint. After that, a transection was conducted with micro-scissors in the anterior cruciate ligament (ACL) in the OA group with a positive anterior drawer sign to confirm the completeness of transection. In the control group, the arthrotomy was conducted while the transection of ACL was not conducted. After surgery, the mice were released from the cages for 30 minutes on a daily basis.The mice were sacrificed by cervical dislocation 12 weeks after surgery and under anesthesia. The knee joint cavity was exposed by separating the patella. The fixed specimens were decalcified for 3 weeks by using 10% ethylenediaminetetraacetic. Upon decalcification, the samples were embedded in paraffin; then the standard serial sections of 3μm each were cut in sagittal plane before the sections were stained by hematoxylin and eosin (H&E), and Alcian blue staining.For immunohistochemical analysis of Klotho and β-catenin expression in articular cartilage of mice, the paraffin-embedded tissues in full-thickness were processed in this study. A blocking serum  was used to incubate the slides for 60 minutes. Then the slides were overlaid with primary antibody against Klotho and β-catenin for 2 hours at room temperatures after being blotted. Subsequently, the sections were added with biotinylated secondary antibodies before they were processed by the streptavidin-biotin staining technique , which is peroxidase-labeled.The first step is to separate chondrocytes from articular cartilage of mice’s knee joints. In addition, the articular cartilage tissues were digested for 30 minutes by using 0.25% trypsin after being cut into small pieces (<1 mm). After this step, digestion using 0.2% Type II collagenase was performed for 3 hours. DMEM/F12 media, antibiotics and 10% fetal bovine serum (FBS) were then used to culture the released cells. After the confluence increased up to 80%, the cells were subjected to cyclic tensile strain for 48 hours at 20% elongation with a 0.1-1 Hz sinusoidal curve indicated by a Flexcell1 FX-5000 Tension System following the user’s instructions . In order to preserve the chondrocyte phenotype, only the cells not exceeding two passages were adopted.Both empty lentiviral vectors and lentiviral vectors encoding Klotho or β-catenin were acquired from Genechem . Lentiviral transfection was conducted by following the manufacturer’s instructions. Green fluorescent protein (GFP) was used to assess the infection efficiency by using flow cytometry. 5μg/ml puromycin  was used to select stably transfected cells after 48 hours.Total protein from cell supernatant, chondrocyte and cartilage was prepared by RIPA buffer, the nuclear protein was isolated by using Nuclear Protein Extraction Kit (Beyotime) strictly in accordance with the Kit instructions, the protein mixtures were separated by gel electrophoresis in sodium dodecyl sulfate-polyacrylamide, which were then translated to nitrocellulose membranes. Briefly, 10% SDS-PAGE was used to extract 15μg plasma proteins from the cartilage tissue and chondrocytes. In addition to that, the proteins were electroblotted onto a polyvinylidene difluoride membrane with a thickness of 0.45 mm , which were then blocked via 5% non-fat dry milk in Tris-buffered saline with Tween 20 for one hour. Incubation of these membranes was then performed at 4 °C overnight with Klotho, Phos-GSK3β (S9), GSK3β, MMP2, MMP9, Wnt1, Wnt4, Wnt7 and β-catenin antibodies at room temperatures for one hour. To visualize antigen-antibody complexes, the enhanced chemiluminescence assay  was applied.Immunoprecipitation lysis buffer  was used to homogenize cartilage tissue on ice before a short step of brief pulses of hand sonication. Following the step of 30-minute incubation on ice, a centrifugation operation was conducted for the lysate and then Protein A/G PLUS-agarose beads were used to preclear the supernatant by incubation at 4 °C. After that, 2μg antibody was used to incubate the precleared supernatant on a rocker at 4 °C for 4 hours against Klitho. The centrifugation was then performed to collect immune-precipitates at 1,000×g for five minutes after adding beads for one hour additionally. Immunoprecipitation lysis buffer was then used to wash the obtained immune-precipitates for 3 times, which were subsequently centrifugated after each wash. 2×sample buffer was used to resuspend and boil the pellet before Western blot was used in the analysis of Wnt1, 4 and 7a by the method as described above.TRIzol reagent (Invitrogen) was used to extract entire RNA from chondrocytes, which was subsequently reversely transcribed into cDNA with the tool of PrimeScript RT reagent kit . SYBR Premix Ex Taq II reagents which were provided by TaKaRa  were used to analyze PCR in a quantitative real-time manner, following the manufacturer’s instructions. 20μL mixture with 2μL cDNA was used to demonstrate PCR results. Specific primers (TaKaRa) were used to amplify Klotho cDNAs. Glyceraldehyde-3-phosphatedehydrogenase (GADPH) was used to normalize gene expression, which was then derived by the 2-DDCT approach. These are the primer sequences: Klotho: forward, 5′-ACTACGTTCAAGTGGACACTACT-3′ and reverse 5′-GATGGCAGAGAAATCAACACAGT-3′; MMP-2: forward, 5′-CAAGTTCCCCGGCGATGTC-3′ and reverse 5′-TTCTGGTCAAGGTCACCTGTC-3′; MMP-9: forward, 5′-CTGGACAGCCAGACACTAAAG-3′ and reverse 5′-CTCGCGGCAAGTCTTCAGAG-3′.ChIP assays were performed essentially as described . NuclearCells were co-transfected by using Lipofectamine 2000 reagent (Life Technologies) with mmp2/mmp9-Luc luciferase reporter constructs (1.0μg) plus shScrambled or shβ-catenin expression plasmid (2.0 μg) (Genechem), respectively. A fixed amount (0.1 μg) of internal control reporter Renilla reniformis luciferase driven under a thymidine kinase promoter was also cotransfected for normalizing the transfection efficiency. Luciferase assay was carried out using a dual luciferase assay system kit according to the manufacturer's protocols (Promega). Relative luciferase activity was reported as fold induction over controls after normalizing for transfection efficiency.Statistics have shown ±SEM of the triplicate values for each experiment. One-way analysis of variance (ANOVA) was used to assess the difference between experiment and control groups. When the P value is <0.05, the difference is significant.Supplementary Figure 1