C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 pathway as a therapeutic target and regulatory mechanism for spinal cord injury

Spinal cord injury involves non-reversible damage to the central nervous system that is characterized by limited regenerative capacity and secondary inflammatory damage.The expression of the C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 axis exhibits significant differences before and after injury.Recent studies have revealed that the C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 axis is closely associated with secondary inflammatory responses and the recruitment of immune cells following spinal cord injury,suggesting that this axis is a novel target and regulatory control point for treatment.This review comprehensively examines the therapeutic strategies targeting the C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 axis,along with the regenerative and repair mechanisms linking the axis to spinal cord injury.Additionally,we summarize the upstream and downstream inflammatory signaling pathways associated with spinal cord injury and the C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 axis.This review primarily elaborates on therapeutic strategies that target the C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 axis and the latest progress of research on antagonistic drugs,along with the approaches used to exploit new therapeutic targets within the C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 axis and the development of targeted drugs.Nevertheless,there are presently no clinical studies relating to spinal cord injury that are focusing on the C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 axis.This review aims to provide new ideas and therapeutic strategies for the future treatment of spinal cord injury.

Mechanokinetics of receptor-ligand interactions in cell adhesion

Receptor-ligand interactions in blood flow are crucial to initiate such biological processes as inflammatory cascade,platelet thrombosis,as well as tumor metastasis.To mediate cell adhesion,the interacting receptors and ligands must be anchored onto two apposing surfaces of two cells or a cell and a substratum,i.e.,two-dimensional（2D）binding,which is different from the binding of a soluble ligand in fluid phase to a receptor,i.e.,three-dimensional（3D） binding.While numerous works have been focused on3 D kinetics of receptor-ligand interactions in the immune system,2D kinetics and its regulations have been less understood,since no theoretical framework or experimental assays were established until 1993.Not only does the molecular structure dominate 2D binding kinetics,but the shear force in blood flow also regulates cell adhesion mediated by interacting receptors and ligands.Here,we provide an overview of current progress in 2D binding and regulations,mainly from our group.Relevant issues of theoretical frameworks,experimental measurements,kinetic rates and binding affinities,and force regulations are discussed.

Homology Model and Ligand Binding Interactions of the Extracellular Domain of the Human α4β2 Nicotinic Acetylcholine Receptor

Addiction to nicotine, and possibly other tobacco constituents, is a major factor that contributes to the difficulties smokers face when attempting to quit smoking. Amongst the various subtypes of nicotinic acetylcholine receptors (nAChRs), the α4β2 subtype plays an important role in mediating the addiction process. The characterization of human α4β2-ligand binding interactions provides a molecular framework for understanding ligand-receptor interactions, rendering insights into mechanisms of nicotine addiction and may furnish a tool for efficiently identifying ligands that can bind the nicotine receptor. Therefore, we constructed a homology model of human α4β2 nAChR and performed molecular docking and molecular dynamics (MD) simulations to elucidate the potential human α4β2-ligand binding modes for eleven compounds known to bind to this receptor. Residues V96, L97 and F151 of the α4 subunit and L111, F119 and F121 of the β2 subunit were found to be involved in hydrophobic interactions while residues S153 and W154 of the α4 subunit were involved in the formation of hydrogen bonds between the receptor and respective ligands. The homology model and its eleven ligand-bound structures will be used to develop a virtual screening program for identifying tobacco constituents that are potentially addictive.

Design of N-11-Azaartemisinins Potentially Active against Plasmodium falciparum by Combined Molecular Electrostatic Potential, Ligand-Receptor Interaction and Models Built with Supervised Machine Learning Methods

N-11-azaartemisinins potentially active against Plasmodium falciparum are designed by combining molecular electrostatic potential (MEP), ligand-receptor interaction, and models built with supervised machine learning methods (PCA, HCA, KNN, SIMCA, and SDA). The optimization of molecular structures was performed using the B3LYP/6-31G* approach. MEP maps and ligand-receptor interactions were used to investigate key structural features required for biological activities and likely interactions between N-11-azaartemisinins and heme, respectively. The supervised machine learning methods allowed the separation of the investigated compounds into two classes: cha and cla, with the properties εLUMO+1 (one level above lowest unoccupied molecular orbital energy), d(C6-C5) (distance between C6 and C5 atoms in ligands), and TSA (total surface area) responsible for the classification. The insights extracted from the investigation developed and the chemical intuition enabled the design of sixteen new N-11-azaartemisinins (prediction set), moreover, models built with supervised machine learning methods were applied to this prediction set. The result of this application showed twelve new promising N-11-azaartemisinins for synthesis and biological evaluation.

Altered expression of stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs)in cancer:will they become a new battlefield for oncotherapy?

The stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs) play pivotal roles in the modulation of Ca^(2+)-regulated pathways from gene transcription to cell apoptosis by driving calcium-dependent signaling processes.Increasing evidence has implicated the dysregulation of STIM-ORAI and IP_3Rs in tumorigenesis and tumor progression.By controlling the activities,structure,and/or expression levels of these Ca^(2+)-transporting proteins,malignant cancer cells can hijack them to drive essential biological functions for tumor development.However,the molecular mechanisms underlying the participation of STIM-ORAI and IP_3Rs in the biological behavior of cancer remain elusive.In this review,we summarize recent advances regarding STIM-ORAI and IP_3Rs and discuss how they promote cell proliferation,apoptosis evasion,and cell migration through temporal and spatial rearrangements in certain types of malignant cells.An understanding of the essential roles of STIM-ORAI and IP_3Rs may provide new pharmacologic targets that achieve a better therapeutic effect by inhibiting their actions in key intracellular signaling pathways.

Overview of the detection methods for equilibrium dissociation constant KD of drug-receptor interaction

Drug-receptor interaction plays an important role in a series of biological effects, such as cell pro- liferation, immune response, tumor metastasis, and drug delivery. Therefore, the research on drug-re- ceptor interaction is growing rapidly. The equilibrium dissociation constant （KD） is the basic parameter to evaluate the binding property of the drug-receptor. Thus, a variety of analytical methods have been established to determine the KD values, including radioligand binding assay, surface plasmon resonance method, fluorescence energy resonance transfer method, affinity chromatography, and isothermal ti- tration calorimetry. With the invention and innovation of new technology and analysis method, there is a deep exploration and comprehension about drug-receptor interaction. This review discusses the differ- ent methods of determining the KD values, and analyzes the applicability and the characteristic of each analytical method. Conclusively, the aim is to provide the guidance for researchers to utilize the most appropriate analytical tool to determine the KD values.

Ligand-Metal Interaction in Transition-Metal Complexes with Tripodal Polyaza Ligands

Five complexes ([Cu(tpc)] 2+ 1, [Cu(tpa)] 2+ 2, [Co(tpa)] 2+ 3, [Fe(tpa)] 2+ 4 and [Mn(tpa)] 2+ 5) (tpc = N, N', N' tris[(2 pyridyl)methyl] 1,4,7 triaza cyclononane(L1) and tpa = tris[2 ((2 pyridyl)methyl imino)ethyl]amine(L2)) were synthesized and characterized. The crystal structures of 1, 3 and 4 were determined by X ray crystallography. Electrochemical investigations over two cupric complexes reveal that 2 has a less negative redox potential than that of 1. Electronic spectra of 2, 3, 4 and 5 show that the distinction in metal ion results in a shift of the ligand based π*←π transition.

A model study on the stacking interaction of phenanthroline ligand with nucleic acid base pairs: An ab initio, MP2 and DFT studies

The stacking of phenanthroline(phen) ligand within base pair sequences is one of the important factors for the stabilization of metalphen complex within DNA. The stacking ability of this ligand has been assessed to deduce the base pair selectivity as well as to identify the favored region of intercalation. Different level of theories have been used to predict the favorable regions for stacking interaction of phen ligand with base pair, but the results of MP2/6-31+G(d,p) is found to be reasonably good for monitoring such interactions.

THE INTRAMOLECULAR AROMATIC-RING STACKING INTERACTION OF MIXED LIGAND PALLADIUM(Ⅱ)COMPLEXES Ⅲ.STUDIES ON THE Pd^(2+)-A-UTP^(4-)SYSTEMS BY HNMR

The intramolecular aromatic-ring stacking interaction of mixed- ligand complex Pd(A)(UTP)^(2-)in the system pd^(2+)-A-UTP^(4-)has been determined by ~1HNMR,where A=1,10-phenanthroline(phen),2,2'-bipyridyl(bpy)and DL- tryptophan(trp^-);UTP^(4-)=uridine 5-triphosphate.The result indicates that it is the partial stacking between the uracil ring of UTP^(4-)and the heterocyclic ring of A that makes H(5),H(6)and H(1')in the UTP^(4-)shift upfield signifi- cantly.Accordingly,the order of aromatic-ring interaction in the mixed- ligand complex has been obtained as follows:Pd(phen)(UTP)^(2-)(?)Pd(bpy)(UTP)^(2-) Pd(trp)(UTP)^(3-).

STUDY ON THE STABILITY AND STACKING INTERACTION EFFECT OF THE TERNARY M(Ⅱ)(ATP)AND PYRIDINE-LIKE LIGANDS

The stabilities of the complexes of three pyridine-like ligands with M(II)(ATP)^(2-) and M(II)(M=Ni,Co)were studied by spectrophotometry and by comparing the stability constants of the ternary complexes with these of the binary complexes.A stacking interaction between the pyridine ring and the purine ring of ATP is indicated.The general existence of the stacking interaction encourages us to interpret the antitumor mechanism of a new class of antitumor drugs.

Designing Artemisinins with Antimalarial Potential, Combining Molecular Electrostatic Potential, Ligand-Heme Interaction and Multivariate Models

Artemisinins tested against W-2 strains of malaria falciparum are investigated with molecular electrostatic potential (MEP), in an attempt to identify key features of the compounds that are necessary for their activities, as well as to investigate likely interactions with the receptor in a biological process and to use that information to propose new molecules. In order to discover the best geometry involving the ligand-receptor complexes (heme) studied and help in the proposition of the new derivatives, molecular simulations of interactions between the most negative charged region around the peroxide and heme locates (the ones around the Fe2+ ion) were carried out. In addition, PCA (principal components analysis), HCA (hierarchical cluster analysis), SDA (stepwise discriminant analysis), and KNN (K-nearest neighbor) multivariate models were employed to investigate which descriptors are responsible for the classification between the higher and lower antimalarial activity of the compounds, and also this information was used to propose new potentially active molecules. The information accumulated in studies of MEP, molecular docking, and multivariate analysis supported the proposal of new structures with potential antimalarial activities. The multivariate models constructed were applied to the new structures and indicated numbers 19 and 20 as the most prominent for syntheses and biological assays.

Role of osteoprotegerin/receptor activator of nuclear factor kappa B/receptor activator of nuclear factor kappa B ligand axis in nonalcoholic fatty liver disease

Concomitantly with the increase in the prevalences of overweight/obesity, nonalcoholic fatty liver disease(NAFLD) has worldwide become the main cause of chronic liver disease in both adults and children. Patients with fatty liver display features of metabolic syndrome(Met S), like insulin resistance(IR), glucose intolerance, hypertension and dyslipidemia. Recently, epidemiological studies have linked obesity, Met S, and NAFLD to decreased bone mineral density and osteoporosis, highlighting an intricate interplay among bone, adipose tissue, and liver. Osteoprotegerin(OPG), an important symbol of the receptor activator of nuclear factor-B ligand/receptor activator of nuclear factor kappa B/OPG system activation, typically considered for its role in bone metabolism, may also play critical roles in the initiation and perpetuation of obesityrelated comorbidities. Clinical data have indicated that OPG concentrations are associated with hypertension, left ventricular hypertrophy, vascular calcification, endothelial dysfunction, and severity of liver damage in chronic hepatitis C. Nonetheless, the relationship between circulating OPG and IR as a key feature of Met S as well as between OPG and NAFLD remains uncertain. Thus, the aims of the present review are to provide the existent knowledge on these associations and to discuss briefly the underlying mechanisms linking OPG and NAFLD.

Neuroprotective effects of receptor imidazoline 2 and its endogenous ligand agmatine

Receptor imidazoline 2 （I2） is one of the imidazoline receptors with high affinity for [^3H]-idazoxan. Receptor I2, being classified into I2A and I28 subtypes, is mainly localized to the outer membrane of mitochondria in liver, kidney and brain. Receptor I2, displaying high similarity of sequence with monoamine oxidase-B （MAO-B）, is structurally related to MAO-B, but the I2 imidazoline binding site （IEBS） with ligand is distinct from the catalytic site of MAO-B. Agmatine is the endogenous ligand of receptor I2. Accumulating evidence have revealed that the activation of receptors I2 may produce neuroprotective effects by increasing expression of glial fibriUary acidic protein （GFAP） in astrocytes, inhibiting activity of MAO, reducing calcium overload in cells. Agmatine exerts neuroprotection against ischemia-hypoxia, injury, glutamateinduced neurotoxicity by activating imidazoline receptors, blocking N-methyl-D-aspartate （NMDA） receptor, inhibiting all isoforms of nitric oxide synthase （NOS）, and selectively blocking the voltage-gated calcium channels （VGCC）. It would be expected that agmatine is one of the potential neuroprotective agents.

Mismatched effects of receptor interacting protein kinase-3 on hepatic steatosis and inflammation in nonalcoholic fatty liver disease

AIM To validate the effects of receptor interacting protein kinase-3(RIP3) deletion in non-alcoholic fatty liver disease(NAFLD) and to clarify the mechanism of action.METHODS Wild-type(WT) and RIP3 knockout(KO) mice werefed normal chow and high fat(HF) diets for 12 wk. The body weight was assessed once weekly. After 12 wk, the liver and serum samples were extracted. The liver tissue expression levels of RIP3, microsomal triglyceride transfer protein, protein disulfide isomerase, apolipoprotein-B, X-box binding protein-1, sterol regulatory element-binding protein-1c, fatty acid synthase, cluster of differentiation-36, diglyceride acyltransferase, peroxisome proliferator-activated receptor alpha, tumor necrosis factor-alpha(TNF-α), and interleukin-6 were assessed. Oleic acid treated primary hepatocytes from WT and RIP3 KO mice were stained with Nile red. The expression of inflammatory cytokines, including chemokine(C-X-C motif) ligand(CXCL) 1, CXCL2, and TNF-α, in monocytes was evaluated.RESULTS RIP3 KO HF diet fed mice showed a significant gain in body weight, and liver weight, liver to body weight ratio, and liver triglycerides were increased in HF diet fed RIP3 KO mice compared to HF diet fed WT mice. RIP3 KO primary hepatocytes also had increased intracellular fat droplets compared to WT primary hepatocytes after oleic acid treatment. RIP3 overexpression decreased hepatic fat content. Quantitative real-time polymerase chain reaction analysis showed that the expression of very-low-density lipoproteins secretion markers(microsomal triglyceride transfer protein, protein disulfide isomerase, and apolipoprotein-B) was significantly suppressed in RIP3 KO mice. The overall NAFLD Activity Score was the same between WT and RIP3 KO mice; however, RIP3 KO mice had increased fatty change and decreased lobular inflammation compared to WT mice. Inflammatory signals(CXCL1/2, TNF-α, and interleukin-6) increased after lipopolysaccharide and pancaspase inhibitor(necroptotic condition) treatment in monocytes. Neutrophil chemokines(CXCL1, and CXCL2) were decreased, and TNF-α was increased after RIP3 inhibitor treatment in monocytes.CONCLUSION RIP3 deletion exacerbates steatosis, and partially inhibits inflammation in the HF diet induced NAFLD model.

MicroRNA-760 acts as a tumor suppressor in gastric cancer development via inhibiting G-protein-coupled receptor kinase interacting protein-1 transcription

BACKGROUND Gastric cancer(GC)has become a serious threat to people's health.Accumulative evidence reveals that dysregulation of numerous microRNAs(miRNAs)has been found during malignant formation.So far,the role of microRNA-760(miR-760)in the development of GC is largely unknown.AIM To measure the expression level of miR-760 in GC and investigate its role in gastric tumorigenesis.METHODS Real-time quantitative polymerase chain reaction and Western blot analysis were used to measure the expression of miR-760 and G-protein-coupled receptor kinase interacting protein-1(GIT1).Cell growth was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)and cell colony formation assays.Apoptosis was assessed by flow cytometric analysis.The relationship between miR-760 and GIT1 was verified by luciferase reporter assay.RESULTS The results showed that the expression of miR-760 was decreased in GC and associated with poor clinical outcomes in GC patients.Furthermore,miR-760 restrained cell proliferation and cell colony formation and induced apoptosis in GC cells.In addition,miR-760 directly targeted GIT1 and negatively regulated its expression in GC.GIT1 was upregulated in GC and predicted a worse prognosis in GC patients.We also found that upregulation of GIT1 weakened the inhibitory CONCLUSION In conclusion,miR-760 targets GIT1 to inhibit cell growth and promote apoptosis in GC cells.Our data demonstrate that miR-760 may be a potential target for the treatment of GC.

Increased Expression of Receptor Activator of Nuclear Factor-κB Ligand in Osteoblasts from Adolescent Idiopathic Scoliosis Patients with Low Bone Mineral Density

Persistent generalized low bone mineral density (BMD) has been reported in patients with adolescent idiopathic scoliosis (AIS).However,the exact mechanisms and causes of the low BMD in AIS patients are largely unknown.The purpose of this study was to examine the relationship between the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) levels in osteoblasts (OBs) from AIS patients with low BMD and with comparison made between the patients and controls.Twenty AIS patients and eight age-matched controls were included in the present study.The BMD of lumbar spine and proximal femur was measured in all subjects.OBs from the cancellous bone of each subject was harvested and primarily cultured.The mRNA and protein expression of RANKL and OPG in OBs was detected by RT-PCR and Western blotting.The results showed BMD was lower in AIS patients than in controls.A significantly higher mRNA and protein expression of RANKL was observed in OBs from AIS patients,while no significant difference was found in the expression of OPG between AIS patients and controls.As a result,RANKL/OPG ratio in patients with AIS was remarkably higher than controls.Our study preliminarily demonstrated expression of RANKL was higher in OBs from AIS patients with low BMD as compared with controls,suggesting the unbalanced RANKL/OPG ratio caused by an over-expression of RANKL in OBs may be responsible for the low BMD in AIS patients.

Science Letters:A synthetic Toll-like receptor 2 ligand decreases allergic immune responses in a mouse rhinitis model sensitized to mite allergen

It has been proposed that activation of Toll-like receptors （TLRs） plays crucial roles in the polarization of adaptive immune responses. A synthetic Toll-like receptor 2 （TLR2） ligand, Pam3CSK4, has been reported to modulate the balance of Th1/Th2 responses. We evaluated the modulation effect of Pam3CSK4 on allergic immune response in a mouse rhinitis model sensitized to house dust mite allergen （HDM）. Mice were sensitized and challenged with Dermatophagoidesfarinae allergen （Der f）, and then the allergic mice were treated by Pam3CSK4. Nasal allergic symptoms and eosinophils were scored. Der f-specific cytokine responses were examined in the splenocytes and bronchoalveolar lavage fluid （BALF）. Serum level of total IgE was also detected. After establishing a mouse allergic rhinitis model with HDM, we have showed that Pam3CSK4 treatment not only ameliorated the nasal allergic symptoms remarkably but also decreased the eosinophils and total inflammation cells in BALF significantly. Analysis of cytokine profile found that IFN-7 released from either BALF or stimulated splenocytes increased markedly in Pam3CSK4-treated mice, while IL-13 decreased significantly. Moreover, serum level of total IgE was significantly lower in Pam3CSK4-treated mice than in the untreated. Thus, in an allergic rhinitis mouse model developed with HDM, Pam3CSK4 was shown to exhibit an antiallergic effect, indicating its potential application in allergic diseases.

Preassembly and ligand-induced restructuring of the chains of the IFN-γ receptor complex： the roles of Jak kinases, Statl and the receptor chains

We previously demonstrated using noninvasive technologies that the interferon-gamma （IFN-γ） receptor complex is preassembled [ 1 ]. In this report we determined how the receptor complex is preassembled and how the ligand-mediated conformational changes occur. The interaction of Statl with IFN-γR1 results in a conformational change localized to IFN- γR1. Jakl but not Jak2 is required for the two chains of the IFN-γ receptor complex （IFN-γR1 and IFN-γR2） to interact; however, the presence of both Jakl and Jak2 is required to see any ligand-dependant conformational change. Two IFN- γR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-γR2 with IFN-γR1. These results agree with a detailed model of the IFN-γ receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.

Interaction of the major inflammatory bowel disease susceptibility alleles in Crohn’s disease patients

AIM:To investigate the interaction of interleukin-23 receptor(IL23R)(rs1004819 and rs2201841),autophagy-related 16-like 1(ATG16L1)(rs2241880), caspase recruitment domain-containing protein 15 (CARD15)genes,and IBD5 locus in Crohn's disease(CD) patients. METHODS:A total of 315 unrelated subjects with CD and 314 healthy controls were genotyped.Interactions and specific genotype combinations of a total of eight variants were tested.The variants of IBD5locus(IGR2198a_1 rs11739135 and IGR2096a_1 rs12521868),CARD15(R702W rs2066845 and L1007fs rs2066847),ATG16L1(rs2241880)and IL23R (rs1004819,rs2201841)genes were genotyped by PCR-RFLP,the G908R(rs2066844)in CARD15 was determined by direct sequencing. RESULTS:The association of ATG16L1 T300A with CD was confirmed[P=0.004,odds ratio(OR)=1.69, 95%CI:1.19-2.41],and both IL23R variants were found to represent significant risk for the disease(P= 0.008,OR=2.05,95%CI:1.20-3.50 for rs1004819 AA;P<0.001,OR=2.97,95%CI:1.65-5.33 for rs2201841 CC).Logistic regression analysis of pairwise interaction of the inflammatory bowel disease (IBD)loci indicated that IL23R,ATG16L1,CARD15 and IBD5(IGR2198a_1)contribute independently to disease risk.We also analysed the specific combina- tions by pair of individual ATG16L1,IL23R rs1004819, rs2201841,IGR2198a_1,IGR2096a_1 and CARD15 genotypes for disease risk influence.In almost all cases,the combined risk of susceptibility pairs was higher in patients carrying two different risk-associated gene variants together than individuals with just one polymorphism.The highest OR was found for IL23R rs2201841 homozygous genotype with combination of positive CARD15 status(P<0.001,OR=9.15,95% CI:2.05-40.74). CONCLUSION:The present study suggests a cumulative effect of individual IBD susceptibility loci.

Effect of Triptolide on Expression of Receptor Activator of Nuclear Factor-κB Ligand in Rat Adjuvant Induced Arthritis

The effect of triptolide （TP） on the expression of receptor activator of nuclear factor-κB ligand （RANKL） and osteoprotegerin （OPG） was explored in rat adjuvant induced arthritis （AA）. AA was induced in Wistar rats. Arthritis rats were treated with TP and methotrexate （MTX） at the onset （day 9） of arthritis. On the peak of arthritis （day 24）, the expression of RANKL and OPG protein in the joints and RANKL mRNA in peripheral blood mononuclear cells （PBMC） was detected. TNF-α and IL-1β levels in peripheral blood were determined. Bone erosion scores were also evaluated. The results showed that bone erosion scores in TP and MTX groups were lower than in AA group （.P〈0.01） ; The expression levels of RANKL in the synovium （P〈0.01） and bone （P〈0.05）, and OPG level in synovium （P〈0.05） were lower in TP group than in AA group （P〈0.05）. In TP group, the expression levels of RANKL mRNA and TNF-α, IL-1β in PBMC were lower than in AA group （all P〈0.01）. It was concluded that TP could inhibit rat adjuvant arthritis bone erosion by suppressing the expression of RANKL.