Midnolin, a Genetic Risk Factor for Parkinson's Disease, Promotes Neurite Outgrowth Accompanied by Early Growth Response 1 Activation in PC12 Cells.

Parkinson's disease (PD) is an age-related progressive neurodegenerative disease. Previously, we identified midnolin (MIDN) as a genetic risk factor for PD. Although MIDN copy number loss increases the risk of PD, the molecular function of MIDN remains unclear. To investigate the role of MIDN in PD, we established monoclonal Midn knockout (KO) PC12 cell models. Midn KO inhibited neurite outgrowth and neurofilament light chain (Nefl) gene expression. Although MIDN is mainly localized in the nucleus, it does not encode DNA-binding domains. We therefore hypothesized that MIDN might bind to certain transcription factors and regulate gene expression. Of the candidate transcription factors, we focused on early growth response 1 (EGR1) because it is required for neurite outgrowth and its target genes are downregulated by Midn KO. An interaction between MIDN and EGR1 was confirmed by immunoprecipitation. Surprisingly, although EGR1 protein levels were significantly increased in Midn KO cells, the binding of EGR1 to the Nefl promoter and resulting transcriptional activity were downregulated as measured by luciferase assay and chromatin immunoprecipitation quantitative real-time polymerase chain reaction. Overall, we identified the MIDN-dependent regulation of EGR1 function. This mechanism may be an underlying reason for the neurite outgrowth defects of Midn KO PC12 cells.

The Relationship Between Quadriceps Femoris Muscle Function and MRI-Derived Water Diffusion and Adipose Tissue Measurements in Young Healthy Males.

BACKGROUND: Water diffusion and adipose tissue in a muscle can be evaluated by MRI. However, determining which quadriceps femoris muscle (QM) characteristics independently predict peak knee extension torque during maximum voluntary isometric contractions (MVICs), individual muscle activity during MVICs and sit-to-stand transitions is unknown. PURPOSE: To determine which QM characteristics predict knee extension muscle strength and individual muscle activity. STUDY TYPE: Prospective, cross-sectional. SUBJECTS: A total of 20 healthy males (aged 22-40 years) with a physical activity level <1 hour/week. FIELD STRENGTH/SEQUENCE: 1.5-T, diffusion-weighted fast spin-echo echo-planar imaging and T1-weighted fast spin-echo sequences. ASSESSMENT: The vastus medialis (VM), vastus lateralis (VL), rectus femoris (RF), and vastus intermedius were segmented in a single axial diffusion-weighted image and T1-weighted image at the right mid-thigh region. λ1-3 and fractional anisotropy (FA), and the percentage of intramuscular adipose tissue (IMAT) were measured. The knee extension peak force during MVICs was measured by a dynamometer, and the torque was calculated at the peak force × length. The ratios of the individual muscle activity to the total muscle (VM, VL, and RF) during MVICs and sit-to-stand transitions were assessed using surface electromyography. STATISTICAL TESTS: Regression analysis was conducted to identify the predictors of peak knee extension torque and of individual muscle activity ratios. A P value < 0.05 was considered statistically significant. RESULTS: The RF λ1 significantly predicted the peak knee extension torque (ß = -0.51). The IMAT percentage of the VM significantly predicted the VM muscle activity ratio during the MVIC and sit-to-stand transition (ß = -0.82 and ß = -0.61, respectively), whereas the ratio of the VM λ1 to the whole QM λ1 significantly predicted the VM muscle activity ratio during the sit-to-stand transitions (ß = 0.35 and ß = 0.46, respectively). DATA CONCLUSION: The RF λ1 may allow to estimate peak knee extension muscle torque, and the VM IMAT and λ1 may predict muscle activity in youth. EVIDENCE LEVEL: 3. TECHNICAL EFFICACY: Stage 3.

The influence of knee varus and valgus on quadriceps muscle activity changes induced by stretching and kneeling.

This study aimed to determine the influence of knee varus (VARUS) and valgus (VALGUS) on the differences in individual quadriceps muscle (QM) activity during knee extension maximum voluntary isometric contractions (MVICs) and sit/stand transitions and on the changes in individual QM activity during sit/stand transitions after QM stretching and kneeling. Ten young healthy males each with VARUS and VALGUS were included. The electromyography signals of the vastus medialis (VM), vastus lateralis, and rectus femoris were recorded during sit/stand transitions before and after rest, stretching, and kneeling and during knee extension MVICs after rest. The individual muscle-to-total muscle activity ratio was assessed. The VARUS group exhibited a significantly higher VM muscle activity ratio in the sit-to-stand and stand-to-sit tasks than in knee extension MVICs (p = 0.004 and p = 0.044, respectively) and a tendency that the VM muscle activity ratio increased in the sit-to-stand task after stretching (p = 0.051), whereas the VALGUS group exhibited no significance. Individuals with VARUS required high VM muscle activity ratios during sit/stand transitions. Future studies should be conducted to determine whether habitual sit-to-stand exercises after QM stretching are effective in preventing knee medial osteoarthritis development in individuals with VARUS.

A risk factor associated with subsequent new vertebral compression fracture after conservative therapy for patients with vertebral compression fracture: a retrospective observational study.

Lumbar BMD and functional recovery in the acute period were independently strongly associated with subsequent new VCF after hospital conservative therapy for patients with new fragility VCFs. Functional recovery was related with age, pain intensity during first month after hospitalization, VCF past history and multiple new VCFs. PURPOSE: This study aimed to determine the factors independently associated with the subsequent vertebral compression fracture (VCF) after hospital conservative therapy in patients with new fragility VCFs and parameters related with functional recovery in the acute period. METHODS: Subsequent VCF (n = 37) was defined as patients who sustained a new VCF within 9 months after new VCF, whereas patients without a new VCF were classified as non-subsequent VCF (n = 179). Logistic regression analysis was performed to determine independent factors associated with the subsequent VCF, including patients' characteristics, past histories, number of new VCFs, bone mineral density (BMD), lumbar sagittal alignment, fractured vertebral body height, spine muscle mass, pain intensity, corset types, medications for osteoporosis and pain relief, recovery ratio of functional independence measure (FIM) and bone union. Correlation coefficients were accessed between the FIM recovery ratio and continuous variable parameters, while intergroup comparisons or analysis of variance was conducted to examine significant differences in the FIM recovery ratio for categorical variable parameters. RESULTS: Lumbar BMD, FIM recovery ratio at the first month after hospitalization and segmental Cobb angle were significantly independently associated with subsequent VCF (odds ratio: 27.8, 9.6 and 1.1, respectively). The FIM recovery ratio was moderately negatively correlated with age and pain intensity and was significantly worse in patients with multiple new VCFs or past history of VCF. CONCLUSIONS: Conservative therapies focused on lumbar BMD, functional recovery and pain relief in the acute period may be useful in preventing subsequent VCF.

Atrophy of individual thigh muscles measured by MRI in older adults with knee osteoarthritis: A cross-sectional study.

BACKGROUND: The characteristics of thigh-muscle cross-sectional area (CSA) in older adults with knee osteoarthritis (KOA) remain controversial. OBJECTIVES: This study aimed to evaluate atrophy of individual thigh muscles in older adults with KOA and to determine which muscle CSA should be measured to detect KOA-related muscle atrophy of the thigh. METHODS: In older adults, individual thigh-muscle CSA measured by 1.5 Tesla MRI was analyzed at 5% intervals of the femoral length (FL) around the mid-thigh between the proximal 25% of the FL and the distal 25%. Participants with KOA grade≤1 and grade≥2 were compared for ratios of quadriceps muscle (QM) CSA to total thigh, individual QM CSA to QM, and individual hamstring (HAM) CSA to HAM at 5% intervals. RESULTS: We included 40 older adults [20 males; mean (SD) age 73.3 (4.7) years; 20 with KOA grade≤1 and 20 with KOA grade≥2]. The ratio of vastus medialis (VM) CSA to QM from the proximal 25% to distal 15% and the ratio of semi-membranosus (SM) CSA to HAM at the distal 10% to 25% were significantly lower with KOA grade≥2 than grade≤1; the effect sizes were 0.34 to 0.67 for VM and 0.40 to 0.60 for SM. The effect sizes were greatest for the ratios of VM CSA to QM at the mid-thigh with 5% intervals and the ratio of SM CSA to HAM at the distal 25%. CONCLUSIONS: The ratio of VM CSA to QM and/or that of SM CSA to HAM were low and were the best indicators to detect KOA-related muscle atrophy of the thigh. However, to detect KOA-related muscle atrophy, the VM CSA ratio should be analyzed in the thigh region around the mid-thigh, whereas the SM CSA ratio should be analyzed in the thigh region at the muscle belly.

Characteristics of individual thigh muscles including cross-sectional area and adipose tissue content measured by magnetic resonance imaging in knee osteoarthritis: a cross-sectional study.

This study aimed to identify the parameters related to the area and adipose tissue content of thigh muscles that are associated with radiographic knee osteoarthritis grade. Fifty patients (mean age ± standard deviation, 73.0 ± 4.5 years) were divided into early osteoarthritis (n = 23) and established osteoarthritis (n = 27) groups based on Kellgren-Lawrence classification. The femorotibial angle was measured from anteroposterior radiographs of the lower limbs. Individual thigh muscle and adipose tissue areas were analyzed using axial T1-weighted magnetic resonance imaging. After intergroup comparison, logistic regression analysis was performed to determine independent parameters associated with established osteoarthritis. Moreover, correlation coefficients were assessed between the left-right differences of osteoarthritis grade and parameters. Established osteoarthritis exhibited a significantly greater femorotibial angle and increased adipose tissue content in the subcutaneous, intermuscle, and intramuscle of the adductor, vastus lateralis, vastus intermedius, as well as a lower vastus medialis area, in comparison to early osteoarthritis. A greater femorotibial angle, increased intermuscular adipose tissue, and a lower vastus medialis area to knee extensor ratio were significantly independently associated with established osteoarthritis (odds ratio 3.2, 1.8, and 2.0, respectively). The left-right differences of femorotibial angle and vastus medialis area were significantly correlated with osteoarthritis grade, whereas adipose tissue content had no significant correlations with osteoarthritis grade. Greater femorotibial angle and lower vastus medialis area were related with higher osteoarthritis grade. Greater intermuscular adipose tissue content was associated with established osteoarthritis; however, in the left-right differences, adipose tissue content was not related with osteoarthritis grade.

Giant Hysteretic Single-Molecule Electric Polarisation Switching above Room Temperature.

Continual progress has been achieved in information technology through unrelenting miniaturisation of the single memory bit in integrated ferromagnetic, ferroelectric, optical, and related circuits. However, as miniaturisation approaches its theoretical limit, new memory materials are being sought. Herein, we report a unique material exhibiting single-molecule electric polarisation switching that can operate above room temperature. The phenomenon occurs in a Preyssler-type polyoxometalate (POM) cluster we call a single-molecule electret (SME). It exhibits all the characteristics of ferroelectricity but without long-range dipole ordering. The SME affords bi-stability as a result of the two potential positions of localisation of a Tb3+ ion trapped in the POM, resulting in extremely slow relaxation of the polarisation and electric hysteresis with high spontaneous polarisation and coercive electric fields. Our findings suggest that SMEs can potentially be applied to ultrahigh-density memory and other molecular-level electronic devices operating above room temperature.

Estimation of individual thigh muscle volumes from a single-slice muscle cross-sectional area and muscle thickness using magnetic resonance imaging in patients with knee osteoarthritis.

PURPOSE: This study aimed to identify the best single-slice anatomical muscle cross-sectional area (CSA) and muscle thickness (MT) on magnetic resonance imaging (MRI) to estimate the overall individual muscle volumes (MVs) of knee extensors and flexors in patients with knee osteoarthritis (KOA). METHODS: Twelve patients (24 legs; 4 men and 8 women) with KOA underwent a 1.5-Tesla axial MRI scan in the femoral region of interest (ROI), between the lesser trochanter and rectus femoris tendon. Individual MVs were calculated by numerical integration based on individual CSAs analyzed at the ROI. The best slice was determined as follows: coefficient of determination ( R2) between MVs measured and those estimated from the femoral length (FL) × CSAs or FL × MTs measured at each 10% interval level of the ROI. These estimation equations were applied for a cross-validation group (24 KOA patients: 12 men and 12 women). RESULTS: Estimated individual MVs of knee extensors and flexors, based on the CSAs at the distal 10% level, significantly correlated with each of the measured individual MVs ( R2: 0.79-0.96, p < 0.05 for all). Similarly, estimated individual knee extensor MVs, based on MTs at the mid-slice, significantly correlated with each of the measured individual MVs ( R2: 0.77-0.84, p < 0.05 for all). The application of the developed regression equation to the cross-validation group did not exhibit any systematic bias. CONCLUSION: These simple methods could be applied in prospective research with a larger number of patients with KOA.

Muscle atrophy and recovery of individual thigh muscles as measured by magnetic resonance imaging scan during treatment with cast for ankle or foot fracture.

PURPOSE: We aimed to longitudinally investigate individual thigh muscle changes using magnetic resonance imaging (MRI) during treatment with cast of ankle or foot fracture. Moreover, we aimed to demonstrate whether measurements of muscle cross-sectional area (CSA) are sensitive to muscle changes, contributing to simpler methods in clinical application . METHODS: Ten patients undergoing treatment with cast of acute ankle or foot fractures were studied. Axial MRI (1.5 T) was conducted around the affected mid-thigh region after the injury (Pre), after maintaining a nonweight-bearing (NWB) period (approximately 28 days), and after finishing rehabilitation (recovery). Regarding individual thigh muscles, the total CSAs corresponding to 40% of the femoral length (FL) and the CSAs at 5% interval of the FL were longitudinally measured. Standardized response means (SRMs) were accessed for sensitivity in the muscle changes. RESULTS: The total CSAs at NWB were significantly lower than those at Pre in vastus lateralis (10.9% ± 5.4%), vastus intermedius (8.4% ± 6.7%), and vastus medialis (11.2% ± 6.9%) ( p < 0.01 for all). In contrast, at recovery, the only significant muscle atrophy relative to that at Pre was observed in the semitendinosus of the proximal 15% and 10% CSAs ( p < 0.01 and p = 0.01, respectively). In all muscles, SRM using a single-slice CSA at or near the muscle belly was high. CONCLUSION: Thigh muscle changes differ according to the variations in individual muscles. CSA measurements at or near the muscle belly are simple methods and sensitive indicators of these muscle changes.

Two New Sandwich-Type Manganese {Mn5}-Substituted Polyoxotungstates: Syntheses, Crystal Structures, Electrochemistry, and Magnetic Properties.

Herein we report two pentanuclear MnII-substituted sandwich-type polyoxotungstate complexes, [{Mn(bpy)}2Na(H2O)2(MnCl)2{Mn(H2O)}(AsW9O33)2]9- and [{Mn(bpy)}2Na(H2O)2(MnCl){Mn(H2O)}2(SbW9O33)2]8- (bpy = 2,2'-bipyridine), whose structures have been obtained by single-crystal X-ray diffraction (SCXRD), complemented by results obtained from elemental analysis, electrospray ionization mass spectrometry, Fourier transform infrared spectroscopy, and thermogravimetric analysis. They consist of two [B-α-XW9O33]9- subunits sandwiching a cyclic assembly of the hexagonal [{Mn(bpy)}2Na(H2O)2(MnCl)2{Mn(H2O)}]9+ and [{Mn(bpy)}2Na(H2O)2(MnCl){Mn(H2O)}2]10+ moieties, respectively, and represent the first pentanuclear MnII-substituted sandwich-type polyoxometalates (POMs). Both compounds have been synthesized by reacting MnCl2·4H2O with trilacunary Na9[XW9O33]·27H2O (X = AsIII and SbIII) POM precursors in the presence of bpy in a 1 M aqueous sodium chloride solution under mild reaction conditions. SCXRD showed that the alternate arrangement of three five-coordinated MnII ions and two six-coordinated MnII ions with an internal Na cation formed a coplanar six-membered ring that was sandwiched between two [B-α-XW9O33]9- (X = AsIII and SbIII) subunits. The results of temperature-dependent direct-current (dc) magnetic susceptibility data indicated ferromagnetic interactions between Mn ions in the cluster. Moreover, alternating-current magnetic susceptibility measurements with a dc-biased magnetic field showed the existence of a ferromagnetic order for both samples. Electrochemistry studies revealed the presence of redox processes assigned to the Mn centers. They are associated with the deposition of material on the working electrode surface, possibly MnxOy, as demonstrated by electrochemical quartz crystal microbalance experiments.

Synthesis of ε-Keggin-Type Cobaltomolybdate-Based 3D Framework Material and Characterization Using Atomic-Scale HAADF-STEM and XANES.

We describe the preparation of ε-Keggin-type cobaltomolybdate-based 3D frameworks with sodium cations, NaH9[ε-CoIIMoV8MoVI4O40CoII2], and their characterization by high-resolution high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and X-ray absorption fine structure (XAFS) spectroscopy. Atomic-scale HAADF-STEM images of ε-Keggin compounds were obtained for the first time, and positions of Mo and Co were confirmed. Furthermore, clear evidence of the presence of a CoO4 tetrahedron was obtained by X-ray absorption near-edge structure (XANES) analysis. Their characterization clearly revealed that ε-Keggin-type cobaltomolybdate units, [ε-CoMo12O40]n-, constructed by a central CoIIO4 tetrahedron and 12 surrounding MoO6 octahedra, are linked with CoII to form 3D frameworks.

Lanthanoid Template Isolation of the α-1,5 Isomer of Dicobalt(II)-Substituted Keggin Type Phosphotungstates: Syntheses, Characterization, and Magnetic Properties.

A new series of heterometallic 3d-4f sandwich type phosphotungstates, [Ln{PCo2W10O38(H2O)2}2](11-) (Ln = Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Tm(III), Yb(III), and Lu(III), denoted 1a-10a, respectively), have been synthesized by a one-pot reaction procedure on reacting the dilacunary K14[P2W19O69(H2O)]·24H2O precursor with Ln(NO3)3·nH2O and Co(NO3)2·6H2O in an aqueous potassium chloride solution. All the compounds were isolated as potassium salts and further characterized with different analytical techniques such as single-crystal X-ray diffraction, Fourier transform infrared spectroscopy, high-resolution electrospray ionization mass spectrometry, elemental analysis by inductively coupled plasma atomic emission spectroscopy, magnetic measurement, and thermogravimetric analysis. Single-crystal X-ray diffraction analysis of the compounds reveals that all these compounds are isostructural and crystallized in the orthorhombic crystal system in space group Iba2. The polyanions contain the α-1,5 isomer of dicobalt-substituted α-Keggin phosphotungstate, which sandwiched lanthanoid cation and formed novel heterometallic dimer species. The temperature-dependent magnetic susceptibilities of 1a, 2a, 4a, and 7a-10a indicate the dominant contribution of the ferromagnetic interaction between Co(II) and Co(II) within the cluster, while the antiferromagnetic interaction between Co(II) and Ln(III) dominates for 3a, 5a, and 6a. The isothermal magnetizations of 1a-10a show a gradual increase in magnetization at low fields and do not reach saturation even at 50 kOe.

Electrical network of single-crystalline metal oxide nanoclusters wired by π-molecules.

In a mixed-valence polyoxometalate, electrons are usually delocalized within the cluster anion because of low level of inter-cluster interaction. Herein, we report the structure and electrical properties of a single crystal in which mixed-valence polyoxometalates were electrically wired by cationic π-molecules of tetrathiafulvalene substituted with pyridinium. Electron-transport characteristics are suggested to represent electron hopping through strong interactions between cluster and cationic π-molecules.

A magnetically isolated cuprate spin-ladder system: synthesis, structures, and magnetic properties.

We synthesized and characterized two magnetically isolated spin ladders, Cu2(CO3)(ClO4)2(NH3)6 (1) and Cu2(CO3)(ClO4)2(H2O)(NH3)5 (2), which are the first examples of carbonate bridging molecular spin ladders. Compounds 1 and 2 form a ladder configuration by stacking a structural unit composed of two Cu(2+) ions and one CO3(2-), where the Cu-O-Cu interactions form the rungs and legs of each ladder and the counter anions (ClO4(-)) occupy the space between the ladders and ensure their magnetic isolation. A S = 1/2 magnetically isolated spin-ladder model with a ladder-rung magnetic interaction J1/k(B) = 364 K (where J is defined as positive for antiferromagnetic interactions) and a ladder-leg magnetic interaction J2/k(B) = 27.4 K accurately predicts the temperature dependence of the molar magnetic susceptibility for 1. The ladder configuration of 2 is similar to that of 1 except that the CO3(2-) is alternately skewed in different directions in the stacked structural unit. Interestingly, this minor structural variation in 2 results in its remarkably different magnetic behavior; the magnetic susceptibility curve of 2 is accurately described by an alternating chain model with J3/k(B) = 7.26 K and J4/k(B) = 4.42 K.

Quick and selective synthesis of Li6[α-P2W18O62]·28H2O soluble in various organic solvents.

Herein we report the synthesis of α-Dawson type POM, Li6[α-P2W18O62]·28H2O, directly from the use of Li2WO4 as the tungstate source. The salt obtained was soluble not only in water but also in a range of polar and non-polar organic solvents, such as benzene.

Bisphosphonate- and statin-induced enhancement of OPG expression and inhibition of CD9, M-CSF, and RANKL expressions via inhibition of the Ras/MEK/ERK pathway and activation of p38MAPK in mouse bone marrow stromal cell line ST2.

Osteoclast differentiation is influenced by receptor activator of the NF-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and CD9, which are expressed on bone marrow stromal cells and osteoblasts. In addition, osteoprotegerin (OPG) is known as an osteoclastogenesis inhibitory factor. In this study, we investigated whether bisphosphonates and statins increase OPG expression and inhibit the expression of CD9, M-CSF, and RANKL in the bone marrow-derived stromal cell line ST2. We found that bisphosphonates and statins enhanced OPG mRNA expression and inhibited the expression of CD9, M-CSF, and RANKL mRNA. Futhermore, bisphosphonates and statins decreased the membrane localization of Ras and phosphorylated ERK1/2, and activated the p38MAPK. This indicates that bisphosphonates and statins enhanced OPG expression, and inhibited the expression of CD9, M-CSF, and RANKL through blocking the Ras/ERK pathway and activating p38MAPK. Accordingly, we believe that its clinical applications will be investigated in the future for the development of osteoporosis therapy.

Macrophage inflammatory protein-1α induces osteoclast formation by activation of the MEK/ERK/c-Fos pathway and inhibition of the p38MAPK/IRF-3/IFN-ß pathway.

Multiple myeloma (MM) is a bone disease that affects many individuals. It was recently reported that macrophage inflammatory protein (MIP)-1α is constitutively secreted by MM cells. MIP-1α causes bone destruction through the formation of osteoclasts (OCs). However, the molecular mechanism underlying MIP-1α-induced OC formation is not well understood. In the present study, we attempted to clarify the mechanism whereby MIP-1α induces OC formation in a mouse macrophage-like cell line comprising C7 cells. We found that MIP-1α augmented OC formation in a concentration-dependent manner; moreover, it inhibited IFN-ß and ISGF3γ mRNA expression, and IFN-ß secretion. MIP-1α increased the expressions of phosphorylated ERK1/2 and c-Fos and decreased those of phosphorylated p38MAPK and IRF-3. We found that the MEK1/2 inhibitor U0126 inhibited OC formation by suppressing the MEK/ERK/c-Fos pathway. SB203580 induced OC formation by upregulating c-fos mRNA expression, and SB203580 was found to inhibit IFN-ß and IRF-3 mRNA expressions. The results indicate that MIP-1α induces OC formation by activating and inhibiting the MEK/ERK/c-Fos and p38MAPK/IRF-3 pathways, respectively, and suppressing IFN-ß expression. These findings may be useful in the development of an OC inhibitor that targets intracellular signaling factors.

Nitrogen-containing bisphosphonate, YM529/ONO-5920, inhibits macrophage inflammatory protein 1 alpha expression and secretion in mouse myeloma cells.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is detected at high concentrations in patients with multiple myeloma, and it is thought to play an important role in the etiology of multiple myeloma and osteolysis. Thus, we investigated whether or not YM529/ONO-5920, a new bisphosphonate, inhibited MIP-1 alpha mRNA expression in, and MIP-1 alpha secretion from, mouse myeloma cells. When the cells were stimulated by lipopolysaccharide, increased MIP-1 alpha mRNA expression and MIP-1 alpha secretion were observed. YM529/ONO-5920 inhibited MIP-1 alpha mRNA expression and MIP-1 alpha secretion in a concentration-dependent manner. A transient increase in the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) and Akt was observed after lipopolysaccharide stimulation. After YM529/ONO-5920 was given, there was no transient increase in the phosphorylation of ERK1/2 or Akt. These results indicated that YM529/ONO-5920 inhibited the expression and secretion of MIP-1 alpha through blocking the signaling pathway of the Ras/mitogen-activated protein kinase kinase/ERK and Ras/phosphatidylinositol-3 kinase/Akt. Accordingly, YM529/ONO-5920 appears to have promise for use in effective future therapy for osteolysis and myeloma cell growth that depends on MIP-1 alpha.

The protein kinase C inhibitor, H7, inhibits tumor cell invasion and metastasis in mouse melanoma via suppression of ERK1/2.

Protein kinase C (PKC) has been shown to be a signal transducer during tumorigenesis, tumor cell invasion, and metastasis. Recent studies have reported that the PKC inhibitor, 7-hydroxystaurosporine, inhibits tumor cell invasion. However, the molecular mechanisms of this inhibition of invasion and metastasis are not well understood. In the present study, we attempt to clarify the mechanism by which H7, a PKC inhibitor, inhibits tumor cell invasion and metastasis in the melanoma cell line B16BL6. It was found that H7 inhibits B16BL6 cell invasion and metastasis. We also observed that H7 inhibits the mRNA expression and protein activities of matrix metalloproteinase (MMP)-1, -2, -9 and MT1-MMP. Furthermore, H7 suppresses phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). However, other signal transduction factors, such as p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase 1/2 (JNK1/2), were unaffected. Moreover, U0126, a MEK1/2 inhibitor, also inhibited B16BL6 cell invasion and metastasis, as well as the mRNA expression and protein activities of MMP-1, -2, -9 and MT1-MMP. This indicates that H7 inhibits signal transduction through the PKC/MEK/ERK pathway, thereby inhibiting B16BL6 cell invasion and metastasis. These results suggest that PKC inhibitors have potential clinical applications in the treatment of tumor cell metastasis.

Macrophage inflammatory protein-1alpha (MIP-1alpha) enhances a receptor activator of nuclear factor kappaB ligand (RANKL) expression in mouse bone marrow stromal cells and osteoblasts through MAPK and PI3K/Akt pathways.

Osteolytic lesions are rapidly progressive during the terminal stages of myeloma, and the bone pain or bone fracture that occurs at these lesions decreases the patients' quality of life to a notable degree. In relation to the etiology of this bone destruction, it has been reported recently that MIP-1alpha, produced in large amounts in myeloma patients, acts indirectly on osteoclastic precursor cells, and activates osteoclasts by way of bone-marrow stromal cells or osteoblasts, although the details of this process remain obscure. In the present study, our group investigated the mechanism by which RANKL expression is induced by MIP-1alpha and the effects of MIP-1alpha on the activation of osteoclasts. RANKL mRNA and RANKL protein expressions increased in both ST2 cells and MC3T3-E1 cells in a MIP-1alpha concentration-dependent manner. RANKL mRNA expression began to increase at 1 h after the addition of MIP-1alpha; the increase became remarkable at 2 h, and continuous expression was observed subsequently. Both ST2 and MC3T3-E1 cells showed similar levels of increased RANKL protein expression at 1, 2, and 3 days after the addition of MIP-1alpha. After the addition of MIP-1alpha, the amount of phosphorylated ERK1/2 and Akt protein expressions showed an increase, as compared to the corresponding amount in the control group. On the other hand, the amount of phosphorylated p38MAPK protein expression showed a decrease from the amount in the control group after the addition of MIP-1alpha. U0126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor) was added to ST2 and MC3T3-E1 cells, and was found to inhibit RANKL mRNA and RANKL protein expression in these cells. When SB203580, a p38MAPK inhibitor, was added, RANKL mRNA and RANKL protein expression were increased in these cells. MIP-1alpha was found to promote osteoclastic differentiation of C7 cells, an osteoclastic precursor cell line, in a MIP-1alpha concentration-dependent manner. MIP-1alpha promoted differentiation into osteoclasts more extensively in C7 cells incubated together with ST2 and MC3T3-E1 cells than in C7 cells incubated alone. These results suggested that MIP-1alpha directly acts on the osteoclastic precursor cells and induces osteoclastic differentiation. This substance also indirectly induces osteoclastic differentiation through the promotion of RANKL expression in bone-marrow stromal cells and osteoblasts. The findings of this investigation suggested that activation of the MEK/ERK and the PI3K/Akt pathways and inhibition of p38MAPK pathway were involved in RANKL expression induced by MIP-1alpha in bone-marrow stromal cells and osteoblasts. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.