Inverse genetics tracing the differentiation pathway of human chondrocytes.

OBJECTIVE: Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) via the forced expression of Yamanaka reprogramming factors. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. This study aimed to clarify the pathways that chondrocytes follow during the reprogramming process. DESIGN: The fate of human articular chondrocytes under reprogramming was investigated through a time-coursed single-cell transcriptomic analysis, which we termed an inverse genetic approach. The iPS interference technique was also employed to verify that chondrocytes inversely return to pluripotency following the proper differentiation pathway. RESULTS: We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a limited population that underwent the silencing of SOX9, a master gene for chondrogenesis, at a specific point during the proper transcriptome transition pathway, could eventually become iPSCs. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway toward cells with a surface zone chondrocyte phenotype. The critical involvement of cellular communication network factors (CCNs) in this process was indicated. The idea that chondrocytes, when reprogrammed into iPSCs, follow the differentiation pathway backward was supported by the successful iPS interference using SOX9. CONCLUSIONS: This inverse genetic strategy may be useful for seeking candidates for the master genes for the differentiation of various somatic cells. The utility of CCNs in articular cartilage regeneration is also supported.

Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.

OBJECTIVE: CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes cartilage regeneration in experimental osteoarthritis (OA) models. However, CCN2 production is very low in articular cartilage. The aim of this study was to investigate whether or not CCN2 was promoted by cultured chondrocytes treated with low-intensity pulsed ultrasound (LIPUS) and to clarify its mechanism. METHODS: Human chondrocytic cell line (HCS)-2/8, rat primary epiphyseal and articular cartilage cells, and Ccn2-deficient chondrocytes that impaired chondrocyte differentiation, were treated with LIPUS for 20 min at 3.0 MHz frequency and 60 mW/cm2 power. Expressions of chondrocyte differentiation marker mRNAs were examined by real-time PCR (RT-PCR) analysis from HCS-2/8 cells and Ccn2-deficient chondrocytes at 30 min and 1 h after LIPUS treatment, respectively. CCN2 production was examined by Western blotting after 5 h of LIPUS treatment. Moreover, Ca2+ influx was measured by using a Fluo-4 probe. RESULTS: The gene expression of chondrocyte differentiation markers and CCN2 production were increased in cultured chondrocytes treated with LIPUS. In addition, Ca2+ influx and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 were increased by LIPUS treatment, and the stability of TRPV4 and BKca channel mRNAs was decreased by siRNA against CCN2. Consistent with those findings, the LIPUS-induced the gene expressions of type II collagen (COL2a1) and Aggrecan (ACAN) observed in wild-type cells were not observed in the Ccn2-deficient chondrocytes. CONCLUSION: These data indicate that chondrocyte differentiation represented by CCN2 production was mediated via MAPK pathways activated by LIPUS-stimulated Ca2+ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes.

Observation of Flat Electron Temperature Profiles in the Lithium Tokamak Experiment.

It has been predicted for over a decade that low-recycling plasma-facing components in fusion devices would allow high edge temperatures and flat or nearly flat temperature profiles. In recent experiments with lithium wall coatings in the Lithium Tokamak Experiment (LTX), a hot edge (>200 eV) and flat electron temperature profiles have been measured following the termination of external fueling. Reduced recycling was demonstrated by retention of ∼60% of the injected hydrogen in the walls following the discharge. Electron energy confinement followed typical Ohmic confinement scaling during fueling, but did not decrease with density after fueling terminated, ultimately exceeding the scaling by ∼200%. Achievement of the low-recycling, hot edge regime has been an important goal of LTX and lithium plasma-facing component research in general, as it has potentially significant implications for the operation, design, and cost of fusion devices.

New millimeter-wave diagnostics to locally probe internal density and magnetic field fluctuations in National Spherical Torus Experiment-Upgrade (invited).

A set of new millimeter-wave diagnostics will deliver unique measurement capabilities for National Spherical Torus Experiment-Upgrade to address a variety of plasma instabilities believed to be important in determining thermal and particle transport, such as micro-tearing, global Alfvén eigenmodes, kinetic ballooning, trapped electron, and electron temperature gradient modes. These diagnostics include a new integrated intermediate-k Doppler backscattering (DBS) and cross-polarization scattering (CPS) system (four channels, 82.5-87 GHz) to measure density and magnetic fluctuations, respectively. The system can access reasonably large normalized wavenumbers kθρs ranging from ≤0.5 to 15 (where ion sound gyroradius ρs = 1 cm and kθ is the binormal density turbulence wavenumber). The system addresses the challenges for making useful DBS/CPS measurements with a remote control of launch polarization (X- or O-mode), probed wavenumber, polarization match of the launch beam with the edge magnetic field pitch angle, and beam steering of the launched beam for wave-vector alignment. In addition, a low-k DBS system consisting of eight fixed frequencies (34-52 GHz) and four tunable frequencies (55-75 GHz) for low-k density turbulence and fast ion physics will be located at a nearby port location. The combined systems cover the near LCFS and pedestal regions (34-52 GHz), the pedestal or mid-radius (50-75 GHz), and core plasmas (82.5-87 GHz).

Design of a low frequency, density profile reflectometer system for the MAST-U spherical tokamak.

Validated and accurate edge profiles (temperature, density, etc.) are vitally important to the Mega Ampere Spherical Tokamak Upgrade (MAST-U) divertor and confinement effort. Density profile reflectometry has the potential to significantly add to the measurement capabilities currently available on MAST-U (e.g., Thomson scattering and Langmuir probes). This work presents the diagnostic requirements, problems, and solutions facing profile reflectometry in spherical tokamaks and MAST-U in particular. Requirements include density measurements near zero electron density in the scrape off layer region, coverage for a broad range of MAST-U plasma parameters, high time (≤10 microseconds) and spatial resolutions (≤1 cm), reliability, and identification of the plasma start frequency.

Impaired glycolytic metabolism causes chondrocyte hypertrophy-like changes via promotion of phospho-Smad1/5/8 translocation into nucleus.

OBJECTIVE: Hypertrophy-like changes are often observed in chondrocytes during the development of osteoarthritis (OA). These changes play a crucial part in the OA-associated cartilage degradation and osteophyte formation. However, the pathogenesis leading to such changes is still unknown. In this study, we investigated the mechanism by which these hypertrophy-like changes are induced from the viewpoint of impaired glycolytic metabolism. METHODS: The effect of sodium fluoride (NaF) on glycolytic metabolism of cultured chondrocytes was confirmed by measurement of intracellular adenosine triphosphate (ATP) production. Translocation of phosphorylated Smad1/5/8 to the nucleus was evaluated by subcellular fractionation and Western blotting. Chondrocyte hypertrophy-like changes were investigated by real-time RT-PCR and Western blot analysis of differentiation markers. RESULTS: ATP production was dose-dependently decreased by NaF in the human chondrocytic cell line HCS-2/8. In addition, both chondrocyte proliferation and differentiation were inhibited, whereas cell death was promoted by treatment with NaF. Interestingly, combinational treatment with NaF and lactate enhanced translocation of phospho-Smad1/5/8 to the nucleus, as well as gene expression of ALP, VEGF, COL10a1, and matrix metalloproteinase13 (MMP13), which were the markers of late mature and hypertrophic chondrocytes. Furthermore, the production of type X collagen and activation of MMP9 were also promoted under the same conditions. CONCLUSIONS: These findings suggest that decreased ATP production by NaF promotes hypertrophy-like changes via activation of phospho-Smad1/5/8 in the presence of lactate. Novel metabolic aspects of OA pathogenesis are indicated herein.

Deletion of thyrotropin receptor residue Asp403 in a hyperfunctioning thyroid nodule provides insight into the role of the ectodomain in ligand-induced receptor activation.

Somatic mutations of the TSH receptor (TSHR) gene are the main cause of autonomously functioning thyroid nodules. Except for mutations in ectodomain residue S281, all of the numerous reported activating mutations are in the TSHR membrane-spanning region. Here, we describe a patient with a toxic adenoma with a novel heterozygous somatic mutation caused by deletion of ectodomain residue Asp403 (Del-D403). Subsequent in vitro functional studies of the Del-D403 TSHR mutation demonstrated greatly increased ligand-independent constitutive activity, 8-fold above that of the wild-type TSHR. TSH stimulation had little further effect, indicating that the mutation produced near maximal activation of the receptor. In summary, we report only the second TSHR ectodomain activating mutation (and the first ectodomain deletion mutation) responsible for development of a thyroid toxic adenoma. Because Del-D403 causes near maximal activation, our finding provides novel insight into TSHR structure and function; residue D403 is more likely to be involved in the ligand-mediated activating pathway than in the ectodomain inverse agonist property.

Neurodegenerative influence of oxidative stress in the retina of a murine model of diabetes.

AIMS/HYPOTHESIS: Diabetic retinopathy is a progressive neurodegenerative disease, but the underlying mechanism is still obscure. Here, we focused on oxidative stress in the retina, and analysed its influence on retinal neurodegeneration, using an antioxidant, lutein. METHODS: C57BL/6 mice with streptozotocin-induced diabetes were constantly fed either a lutein-supplemented diet or a control diet from the onset of diabetes, and their metabolic data were recorded. In 1-month-diabetic mice, reactive oxygen species (ROS) in the retina were measured using dihydroethidium and visual function was evaluated by electroretinograms. Levels of activated extracellular signal-regulated kinase (ERK), synaptophysin and brain-derived neurotrophic factor (BDNF) were also measured by immunoblotting in the retina of 1-month-diabetic mice. In the retinal sections of 4-month-diabetic mice, histological changes, cleaved caspase-3 and TUNEL staining were analysed. RESULTS: Lutein did not affect the metabolic status of the diabetic mice, but it prevented ROS generation in the retina and the visual impairment induced by diabetes. ERK activation, the subsequent synaptophysin reduction, and the BDNF depletion in the diabetic retina were all prevented by lutein. Later, in 4-month-diabetic mice, a decrease in the thickness of the inner plexiform and nuclear layers, and ganglion cell number, together with increase in cleaved caspase-3- and TUNEL-positive cells, were avoided in the retina of lutein-fed mice. CONCLUSIONS/INTERPRETATION: The results indicated that local oxidative stress that has a neurodegenerative influence in the diabetic retina is prevented by constant intake of a lutein-supplemented diet. The antioxidant, lutein may be a potential therapeutic approach to protect visual function in diabetes.

Induction of lymphokine-activated killer-like cells by cancer chemotherapy.

Natural cell-mediated cytotoxicity against NK-resistant target tumor cells was found in the peripheral blood of tumor-bearing patients approximately 1 mo after combined chemotherapy. The recognition specificity of these effector cells was broad and had no restriction. From the experiments of negative selection with mAbs and complements, these newly developed killer cells after chemotherapy were thought to be LAK-like cells. Contribution of these LAK-like cells to the mechanism of action of anticancer drugs remains to be clarified.

Chromosome evolution in the lizard genus Gekko (Gekkonidae, Squamata, Reptilia) in the East Asian islands.

The lizard genus Gekko consists of over 30 species distributed in Asia and Oceania. From the insular region of East Asia including Japan and Taiwan, 9 species (G. hokouensis, G. japonicus, G. shibatai, G. tawaensis, G. vertebralis,G. yakuensis, and 3 undescribed species) are currently recognized. We made karyological analyses for all these species. Their karyotypes invariably consisted of 2N = 38 chromosomes, but exhibited considerable variation in fundamental number (ranging from 56-62). Substantial chromosomal variation was detected even among populations of a morphologically relatively uniform species, G. hokouensis. Populations of G. hokouensis from the central and northern Ryukyus exhibited prominent female heteromorphic (i.e., ZW type) sex chromosomes. Populations of the southern Ryukyus exclusive of Yonagunijima also had ZW sex chromosomes, whose heteromorphisms were, however, much less prominent. The other G. hokouensis populations including the topotypic continental representatives and the population from Yonagunijima of the southern Ryukyus exhibited no sex chromosome heteromorphism at all. These results strongly suggest that G. hokouensis in the current taxonomic definition actually includes more than 2 species. The process of chromosomal evolution in the East Asian Gekko is hypothesized.

Effect of transforming growth factor-beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro. MATERIAL AND METHODS: Cells were isolated from normal periodontal tissues and cultured in Dulbecco's modified Eagle's minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco's modified Eagle's minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription-polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy. RESULTS: In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization. CONCLUSION: The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue.

Extent of hypoechogenic area in the thyroid is related with thyroid dysfunction after subacute thyroiditis.

OBJECTIVE: To gain an insight into risk factors for hypothyroidism after subacute thyroiditis (SAT), we examined the correlation between initial laboratory and ultrasonographic findings and sequential thyroid dysfunction among treatment modalities. PATIENTS: We reviewed retrospectively the medical records of 252 patients (26 men and 226 women) with SAT who consecutively visited our thyroid clinic at Kuma Hospital for at least 6 months from 1996 through 2004. RESULTS: Throughout the course, 135 patients (53.6%) developed transient or permanent hypothyroidism. Levels of TSH were most often elevated (greater than 5 IU/ml) 2 months after SAT onset regardless of treatment, and 97.0% of patients who showed transient or permanent hypothyroidism clustered within 6 months from onset. During follow-up, patients treated with prednisone (PSL) were more likely to have normal thyroid function than patients not treated or those receiving anti-inflammatory drug therapy. In patients who developed hypothyroidism with PSL treatment or without treatment, the rates of bilateral hypoechogenic areas (HEA) were 6-fold higher than those of unilateral HEA. Moreover, permanent hypothyroidism occurred in 5.9% of patients, and all patients with permanent hypothyroidism presented initially with bilateral HEA and had consequently small thyroid size with or without abnormal autoimmunity. CONCLUSIONS: The rates of thyroid dysfunction after SAT were significantly lower in patients receiving PSL. Extent of HEA in the thyroid, but not laboratory findings, may be a possible marker for developing thyroid dysfunction after SAT.

Interobserver agreement in endoscopic evaluation of reflux esophagitis using a modified Los Angeles classification incorporating grades N and M: a validation study in a cohort of Japanese endoscopists.

The Los Angeles classification system is the most widely employed criteria associated with the greatest interobserver agreement among endoscopists. In Japan, the Los Angeles classification system has been modified (modified LA system) to include minimal changes as a distinct grade of reflux esophagitis, rather than as auxiliary findings. This adds a further grading M defined as minimal changes to the mucosa, such as erythema and/or whitish turbidity. The modified LA system has come to be used widely in Japan. However, there have been few reports to date that have evaluated the interobserver agreement in diagnosis when using the modified LA classification system incorporating these minimal changes as an additional grade. A total of 100 endoscopists from university hospitals and community hospitals, as well as private practices in the Osaka-Kobe area participated in the study. A total of 30 video clips of 30-40 seconds duration, mostly showing the esophagocardiac junction, were created and shown to 100 endoscopists using a video projector. The participating endoscopists completed a questionnaire regarding their clinical experience and rated the reflux esophagitis as shown in the video clips using the modified LA classification system. Agreement was assessed employing kappa (kappa) statistics for multiple raters. The kappa-value for all 91 endoscopists was 0.094, with a standard error of 0.002, indicating poor interobserver agreement. The endoscopists showed the best agreement on diagnosing grade A esophagitis (0.167), and the poorest agreement when diagnosing grade M esophagitis (0.033). The kappa-values for the diagnoses of grades N, M, and A esophagitis on identical video pairs were 0.275-0.315, with a standard error of 0.083-0.091, indicating fair intraobserver reproducibility among the endoscopists. The study results consistently indicate poor agreement regarding diagnoses as well as fair reproducibility of these diagnoses by endoscopists using the modified LA classification system, regardless of age, type of practice, past endoscopic experience, or current workload. However, grade M reflux esophagitis may not necessarily be irrelevant, as it may suggest an early form of reflux disease or an entirely new form of reflux esophagitis. Further research is required to elucidate the pathophysiological basis of minimal change esophagitis.

[An adult case of double aortic arch].

Reports of surgical treatment are rare in adults with double aortic arch. We present such a case of a 15-year-old male who presented with dysphagia and dyspnea. Diagnosis was made by magnetic resonance imaging (MRI), showing a dominant right arch. The patient underwent divisions of left aortic arch and ligamentum arteriosum, and further dissections around esophagus and trachea. After the operation, both dysphagia and dyspnea were disappeared completely. By reviewing literatures in adult cases, cautions in surgical procedure were discussed in this article.

Millimeter-wave interferometry and far-forward scattering for density fluctuation measurements on LTX-ß.

The λ ≈ 1 mm (f = 288 GHz) interferometer for the Lithium Tokamak Experiment-ß (LTX-ß) will use a chirped-frequency source and a centerstack-mounted retro-reflector mirror to provide electron line density measurements along a single radial chord at the midplane. The interferometer is unique in the use of a single source (narrow-band chirped-frequency interferometry) and a single beam splitter for separating and recombining the probe and reference beams. The current work provides a documentation of the interferometry hardware and evaluates the capabilities of the system as a far-forward collective scattering diagnostic. As such, the current optical setup is estimated to have a detection range of 0.4 ≲ k ⊥ ≲ 1.7 cm-1, while an improved layout will extend the upper k ⊥ limit to ∼3 cm-1. Measurements with the diagnostic on LTX are presented, showing interferometry results and scattered signal data. These diagnostics are expected to provide routine measurements on LTX-ß for high frequency coherent density oscillations (e.g., Alfvénic modes during neutral beam injection) as well as for broadband turbulence.

Hypoxic regulation of stability of connective tissue growth factor/CCN2 mRNA by 3'-untranslated region interacting with a cellular protein in human chondrosarcoma cells.

Connective tissue growth factor (CTGF/CCN2) can be induced by various forms of stress such as exposure to high glucose, mechanical load, or hypoxia. Here, we investigated the molecular mechanism involved in the induction of ctgf/ccn2 by hypoxia in a human chondrosarcoma cell line, HCS-2/8. Hypoxia increased the ctgf/ccn2 mRNA level by altering the 3'-untranslated region (UTR)-mediated mRNA stability without requiring de novo protein synthesis. After a series of extensive analyses, we eventually found that the cis-repressive element of 84 bases within the 3'-UTR specifically bound to a cytoplasmic/nuclear protein. By conducting a UV crosslinking assay, we found the cytoplasmic/nuclear protein to be a 35 kDa molecule that bound to the cis-element in a hypoxia-inducible manner. These results suggest that a cis-element in the 3'-UTR of ctgf/ccn2 mRNA and trans-factor counterpart(s) play an important role in the post-transcriptional regulation by determining the stability of ctgf/ccn2 mRNA.

Impaired oxygen utilization. A new mechanism for the hepatotoxicity of ethanol in sub-human primates.

The role of oxygenation in the pathogenesis of alcoholic liver injury was investigated in six baboons fed alcohol chronically and in six pair-fed controls. All animals fed alcohol developed fatty liver with, in addition, fibrosis in three. No evidence for hypoxia was found, both in the basal state and after ethanol at moderate (30 mM) or high (55 mM) levels, as shown by unchanged or even increased hepatic venous partial pressure of O2 and O2 saturation of hemoglobin in the tissue. In controls, ethanol administration resulted in enhanced O2 consumption (offset by a commitant increase in splanchnic blood flow), whereas in alcohol fed animals, there was no increase. At the moderate ethanol dose, the flow-independent O2 extraction, measured by reflectance spectroscopy on the liver surface, tended to increase in control animals only, whereas a significant decrease was observed after the high ethanol dose in the alcohol-treated baboons. This was associated with a marked shift in the mitochondrial redox level in the alcohol-fed (but not in control) baboons, with striking rises in splanchnic output of glutamic dehydrogenase and acetaldehyde, reflecting mitochondrial injury. Increased acetaldehyde, in turn, may aggravate the mitochondrial damage and exacerbate defective O2 utilization. Thus impaired O2 consumption rather than lack of O2 supply characterizes liver injury produced by high ethanol levels in baboons fed alcohol chronically.

Usefulness of the long-acting insulin analogue glargine in basal-bolus therapy for Japanese children and adolescents with type 1 diabetes mellitus.

The aim of this study was to evaluate the efficacy of long-acting insulin analogue glargine (G) changing from NPH in basal-bolus therapy for Japanese children and adolescents with type 1 diabetes mellitus (DM1). Thirty patients (11 M, 19 F) with DM1 aged 13.3 +/- 4.5 years were included in the study. Mean fasting blood glucose level was significantly decreased (baseline: 142.5 +/- 39.3 vs 127.1 +/- 24.0, 129.0 +/- 29.1, 121.1 +/- 26.0 mg/dl at 3, 6, 12 months, respectively, p <0.01), and mean HbA(1c) was significantly decreased (baseline: 8.06 +/- 0.85 vs 7.69 +/- 0.89, 7.57 +/- 0.93, 7.36 +/- 0.95%, at 3, 6, 12 months, respectively, p <0.01) after changing to G from NPH. Severe hypoglycemia rarely occurred during the study period. In conclusion, basal-bolus therapy using G resulted in improved overall glycemic control with a low risk of severe hypoglycemia in Japanese pediatric patients with DM1.

A frequency-modulated continuous-wave reflectometer for the Lithium Tokamak Experiment.

The frequency-modulated continuous-wave reflectometer on LTX (Lithium Tokamak Experiment) and the data analysis methods used for determining electron density profiles are described. The diagnostic uses a frequency range of 13.1-33.5 GHz, for covering a density range of 0.21-1.4×1013 cm-3 (in O-mode polarization) with a time resolution down to 8 µs. The design of the diagnostic incorporates the concept of an "optimized" source frequency sweep, which minimizes the large variation in the intermediate frequency signal due to a long dispersive transmission line. The quality of the raw data is dictated by the tuning characteristics of the microwave sources, as well as the group delay ripple in the transmission lines, which can generate higher-order nonlinearities in the frequency sweep. Both effects are evaluated for our diagnostic and best practices are presented for minimizing "artifacts" generated in the signals. The quality of the reconstructed profiles is also improved using two additional data analysis methods. First, the reflectometer data are processed as a radar image, where clutter due to echoes from the wall and backscattering from density fluctuations can be easily identified and removed. Second, a weighed least-squares lamination algorithm POLAN (POLynomial ANalysis) is used to reconstruct the electron density profile. Examples of density profiles in LTX are presented, along with comparisons to measurements from the Thomson scattering and the λ = 1 mm interferometer diagnostics.

Multiple activation of mitogen-activated protein kinases by purified independent CCN2 modules in vascular endothelial cells and chondrocytes in culture.

CCN2 consists of 4 distinct modules that are conserved among various CCN family protein members. From the N-terminus, insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C repeat (VWC), thrombospondin type 1 repeat (TSP1) and C-terminal cysteine-knot (CT) modules are all aligned tandem therein. The multiple functionality of CCN2 is thought to be enabled by the differential use of these modules when interacting with other molecules. In this study, we independently prepared all 4 purified module proteins of human CCN2, utilizing a secretory production system with Brevibacillus choshinensis and thus evaluated the cell biological effects of such single modules. In human umbilical vascular endothelial cells (HUVECs), VWC, TSP and CT modules, as well as a full-length CCN2, were capable of efficiently activating the ERK signal transduction cascade, whereas IGFBP was not. In contrast, the IGFBP module was found to prominently activate JNK in human chondrocytic HCS-2/8 cells, while the others showed similar effects at lower levels. In addition, ERK1/2 was modestly, but significantly activated by IGFBP and VWC in those cells. No single module, but a mixture of the 4 modules provoked a significant activation of p38 MAPK in HCS-2/8 cells, which was activated by the full-length CCN2. Therefore, the signals emitted by CCN2 can be highly differential, depending upon the cell types, which are thus enabled by the tetramodular structure. Furthermore, the cell biological effects of each module on these cells were also evaluated to clarify the relationship among the modules, the signaling pathways and biological outcomes. Our present results not only demonstrate that single CCN2 modules were potent activators of the intracellular signaling cascade to yield a biological response per se, while also providing new insight into the module-wise structural and functional relationship of a prototypic CCN family member, CCN2.