State-of-the-Art Sensors Technology in Spain 2013, this paper also reviews the most important flatness sensors designed and developed for the steelmaking industry in Spain.Shape is a key characteristic to determine the quality of outgoing flat-rolled products in the steel industry. It is greatly influenced by flatness, a feature to describe how the surface of a rolled product approaches a plane. Flatness is of the utmost importance in steelmaking, since it is used by most downstream processes and customers for the acceptance or rejection of rolled products. Flatness sensors compute flatness measurements based on comparing the length of several longitudinal fibers of the surface of the product under inspection. Two main different approaches are commonly used. On the one hand, most mechanical sensors measure the tensile stress across the width of the rolled product, while manufacturing and estimating the fiber lengths from this stress. On the other hand, optical sensors measure the length of the fibers by means of light patterns projected onto the product surface. In this paper, we review the techniques and the main sensors used in the steelmaking industry to measure and quantify flatness defects in steel plates, sheets and strips. Most of these techniques and sensors can be used in other industries involving rolling mills or continuous production lines, such as aluminum, copper and paper, to name a few. Encompassed in the special issue,  This characteristic is essential in modern high-speed and high-volume finishing lines, where flat-rolled products must fulfill tight shape tolerances. Furthermore, shape is a main feature used by customers to accept or reject incoming material provided by steel suppliers. Thus, new operating strategies aiming to reduce shape defects and yield losses are continuously being proposed ,2 for stFrom a mathematical point of view, flatness can be defined as the degree to which all the elements of a surface approach a plane . In flatModern rolling mills combine higher rolling speeds, larger reductions of the incoming workpiece, harder steel grades and thinner rolled output products. Thus, to ensure high quality levels in terms of flatness and defect-free surfaces, knowledge of friction and lubrication in the roller gap becomes critical . Some seUsually, flatness tolerances are determined depending on the use of the manufactured product and on the requirements imposed by the customer. To achieve the desired flatness, a flatness control system—which drives the mechanical and thermal actuators to adjust the shape of the product—is used. Mechanical actuators adapt the roller gap, the bending and the tilting of each rolling mill stand , whereasThe improvement of shape quality in the steelmaking industry began in the early 1960s with the improvement of width and thickness accuracy. In those days, flatness measurements were performed off-line and manually by humans, until the first automated devices were developed in the late 1960s. The first automated flatness sensor for steel rolling mills was delivered in 1967, as ABB (prior ASEA)claims. Early automated flatness sensors were contact devices based on mechanical principles. They used strength or pressure transducers integrated into a deflector roller to measure the radial force applied by the product as it moved along the manufacturing line. The principle of measurement was based on the fact that radial force along the rolling direction is directly proportional to the tension profile in the rolled product, that is, product tension plus residual stress, assuming the shear stress is negligible. In the mid-1980s, non-contact flatness sensors based on optical principles started to be developed. They mainly used triangulation and structured light to compute the 3D surface of the product under inspection. Both mechanical and optical principles are currently used in modern flatness sensors for the steelmaking industry. The main advantage of mechanical systems is that they can be installed in very harsh atmospheres, where optical systems will be likely to fail. However, they cannot be used with very thick or very hot rolled products, because the pressure sensors might be damaged. Conversely, optical flatness sensors provide the advantage of non-contact sensors for high quality surface manufacturing. In the steelmaking industry, high quality surfaces are referred to as technically free from manufacturing cracks ,12. AlthModern automated flatness sensors in the steelmaking industry are required to provide on-line flatness measurements. First, these measurements are required as feedback for flatness control systems see . Second,State-of-the-Art Sensors Technology in Spain 2013, Section 4 reviews the most important flatness measurement sensors designed and developed for the steelmaking industry in Spain in the last three decades. Finally, Section 5 shows a comparison of the main features of all the flatness sensors described in Sections 3 and 4, as well as the final impressions of this review.This paper reviews the technologies and applications used for on-line flatness measurement in the steelmaking industry to measure and quantify flatness defects in steel plates, sheets and strips. Most of them can be used in other industries where rolling mills or continuous production lines are used, such as aluminum, copper and paper. The rest of this paper is organized as follows. Section 2 describes how flatness defects are generated in steel, flat-rolled products in rolling mills and production lines, the main types of flatness defects and how they can be quantitatively evaluated. Section 3 describes the main techniques used in on-line flatness measurement systems in the steelmaking industry and the main flatness sensors developed based on each technique that are commercially available. Since this paper is encompassed in the special issue, 2.Steel, flat-rolled products—such as plates, sheets and strips—are manufactured from large, hot slabs that are passed through several rolling stands in a rolling mill, reducing their thickness and increasing their length. The rolling process can be symmetric or asymmetric. Symmetric rolling applies equal rolling conditions at the roll-slab interface by keeping the roller diameters, the roller speeds, the roller-slab friction and the slab entry between rollers symmetrical with respect to the middle plane of the slab. The output of symmetric rolling is straight, flat-rolled products. On the other hand, asymmetric rolling can arise for several reasons, such as difference of the surface roughness or diameters of the rollers or when rolling bimetallic slabs. The output of asymmetric rolling is curly, flat-rolled products .In the steel industry, it is well known that when the thickness profile across the width of the slab is constant, the outgoing flat-rolled product will have non-flat edges, since the edges of the slab can expand to the free space of the roller gap. To compensate for this effect, the rollers in the rolling stands are bent to laminate the slab with a predefined thickness profile, which maximizes the likelihood of the rolled product being perfectly flat . In addiA quantitative evaluation of flatness defects in flat-rolled products is commonly carried out considering the rolled product divided as multiple, adjacent longitudinal fibers, as shown in Waves in flat-rolled products may appear in different sections, depending on the location of the fiber elongations. When the length of the fibers at the edges of the rolled product is longer than the length of the fibers at the center, the rolled product is said to have a flatness defect, called wavy edges, as shown in refL, are used to report its flatness. Typically, refL takes values from 2.5 to 10 meters.Flat-rolled products in the steel industry are considerably long, especially strips that can be several hundreds of meters long. Thus, longitudinal sections of the rolled product having a reference length, jF is the I-unit index of the fiber, j, and ΔjL/refL is the elongation of the fiber, j, compared to the reference fiber, ref. The factor used by the I-unit index (105) made it equivalent to 10 μm/m.Usually, flatness defects in the steelmaking industry are quantitatively evaluated using a dimensionless metric, called I-units , computejF is the I-unit index of the fiber, j, ΔjL/refL is the elongation of the fiber, j, compared to the reference fiber, ref,jσ is the longitudinal tensile stress of the fiber, j,mσ is the mean longitudinal stress across the product width and E is the modulus of elasticity of the steel product.As shown above, flatness measurements of a rolled product in the steelmaking industry can be computed based on length measurements taken over several longitudinal sections of the product. However, there are some factors that may make these length measurements inaccurate in some manufacturing processes: the tension of the product, fast tension variations, the deflection angle, the temperature profile and the product thickness. When flat-rolled products are processed with no tension, or under low tension levels, the waves of the product are not modified, and the flatness defects can be directly seen. In this case, flatness defects are said to be manifest. Conversely, when the products are processed under high tension levels, this tension may mask some flatness defects or the real magnitude of these defects. In this case, flatness defects are latent, that is, they cannot be seen directly, but they will appear in the product when the manufacturing tension is released. The term, critical buckling stress, is often used to refer to the stress level where latent defects turn into manifest defects. Therefore, when the products are processed under high tension levels, a comparison between the tensile stress of the fibers is preferred to compute their lengths. The flatness index of a fiber of the product can be computed from the tensile stress using the derived formula of Hooke's law shown in Since the I-unit index expresses the relation between the length of a fiber compared to a reference fiber, a flatness profile can be obtained by computing the I-unit index for each fiber, that is, the distribution of the relative elongation of the fibers. F1+n-c is the symmetrical flatness and L1, cL and nL are the length of the fiber at one side, at the center and at the other side of the product, respectively. On the other hand, asymmetrical flatness measurements can be used to detect irregularities in the tilting of the rollers and can be computed as shown in F1-n is the asymmetrical flatness.Once flatness measurements have been computed for all the fibers of the rolled product, two more specific measurements can be provided from them, symmetrical and asymmetrical flatness. Symmetrical flatness measurements can be used to describe flatness defects caused by bending irregularities in the rolling stand and can be computed as shown in jH is the peak-to-peak wave amplitude of the fiber, j, and jL is the wavelength of the fiber, j. In practice, on-line measurements recorded under tension cannot always be correlated with the values measured off-line with the aid of other instruments [2 of tension applied by the production line and one I-unit per °C of temperature variation of the rolled product. Thus, flatness measurements taken in two different locations of the steelmaking plant may vary, and such correlation could be very hard to find.Sometimes, comparisons between the flatness measurements provided by an on-line flatness sensor and the flatness measurements computed using off-line methods of the same rolled product are required. However, it is very difficult to take measurements of the length of several longitudinal fibers of the rolled product off-line. Furthermore, it is not possible to measure the tensile stress across the width of the product, since no tension is applied. Therefore, off-line flatness measurements are usually carried out assuming sine-shaped waves of the rolled product, and after measuring their wavelength and amplitude, flatness measurements are computed using truments . After b3.i) whether or not the sensor is required to be in contact with the surface of the product under inspection; and (ii) the physical principle used by the sensor. Using the first criterion, flatness sensors are classified into contact and non-contact devices, whereas using the second criterion, they are classified into mechanical or optical devices. In this paper, the first criterion is used, since it is the most commonly used in the steelmaking industry.Usually, flatness sensors provide flatness measurements based on a two-stage operation. In the first stage, topographical information about the surface of the product under inspection is obtained. This topographical information consists of the length of several longitudinal sections, or fibers, of the rolled product. In the second stage, flatness measurements are computed from this information. Typically, flatness sensors are classified based on the technique used in the first stage. Two main criteria can be used:  in 1967 [® in 1954 [The first commercially available, roll-based flatness measurement system Stressometer in 1967 . It was  in 1954 , detectsThe second well-known technology for roll-based flatness measurement systems was developed by VDEh-Betriebsforschungsinstitut (BFI) . This teRoll-based flatness measurement sensors provide accurate measurements that are consistent when the tension applied to the rolled product varies in the manufacturing process. The main drawback of these systems is that they are intrusive measurement devices, and thus, although they can use very well-treated surfaces, they may scratch or stretch the surface of the product under inspection. Furthermore, contact flatness sensors cannot be used to measure the flatness of steel plates, since they are not in tension during manufacturing. They move along the processing line, due to the effect of rolling stands or motorized rollers underneath them. In all these scenarios, non-contact flatness sensors are preferred.Usually, roll-based flatness sensors are not suitable for hot rolling mills. The high temperatures of the rolled product may cause rapid wear and tear of the roller, reducing its operational life sharply and increasing the maintenance time and the amount of spare devices required for the production line. A roll-based flatness sensor for flat-rolled products at high temperatures has been patented by Clecim  . The rolRoll-based flatness sensors are not suitable either to measure thick rolled products (above one or 2 mm). In these products, the critical buckling stress is quite high, and the tension distribution measured with a roll-based sensor would provoke too high of a contact stress between the roller and the product. Furthermore, the deflection angle would provoke plastic deformation in thick products when the product wraps on the roller. Thus, these manifest flatness defects must be measured using another technique. On the other hand, the critical buckling stress is low in thin-rolled products, and any small heterogeneity of internal stress can be masked by the tension in the manufacturing line. Thus, roll-based flatness sensors are used in this scenario to measure the latent flatness defects.As mentioned above, roll-based sensors are the most commonly used devices for flatness measurement in cold rolling in the steelmaking industry. This type of sensor provides flatness measurements that match with predictions provided by modern numerical simulations . However3.2.Optical methods are widely used for contactless shape measurement in industry to speed up and ensure product development and manufacturing quality . SurfaceOptical sensors in industry tend to use controlled lighting methods, that is, active methods, when they are installed in hostile environments to avoid external noise. The most commonly used method for contactless flatness measurement in the steelmaking industry is active triangulation. Active optical triangulation systems require a controlled light source, which projects a light pattern onto the surface of the product. Flatness defects provoke variations in the surface of the product, and thus, variations in the projected pattern. These variations are imaged by a sensor, typically a charge-coupled device (CCD) camera, and then they are processed to compute the lengths of the fibers of the surface. Although white-light projectors can be used, laser projectors are usually preferred as the active light sources for these systems, given their high spectral radiance, which allows one to reduce the sensor exposure time per measurement.Since optical flatness sensors are non-contact devices, they cannot measure the tensile stress across the width of the rolled product while manufacturing. Thus, optical flatness sensors are only able to measure manifest flatness defects, as opposed to roll-based flatness sensors that can measure latent flatness defects. Therefore, optical-based flatness sensors require low tension to provide accurate measurements. Furthermore, some optical flatness sensors—mainly those based on active triangulation—may not be suitable for products featuring very bright surfaces.3.2.1.Primitive flatness sensors based on active optical triangulation used discrete light points. The points were projected onto the surface of the product to be inspected, and their projections were acquired by linear-sensor cameras, as shown in The first commercially available system based on this technology, Rometer, was developed by IRM in the mid-1980s ,39. It uThe evolution of laser technology allowed flatness sensors to use line projectors instead of discrete-point projectors. In these sensors, a matrix camera is used to image the laser stripe projected onto the product surface, as shown in jL is the computed approximation of the length of the fiber, j, j, in the i-th sample, it is the time stamp where the sample, i, was taken, iv is the speed of the product movement and n is the total amount of samples to compute the length of the fiber. Flatness sensors based on optical triangulation compute the length of each fiber based on an integration technique that uses the height variations of the fiber between consecutive samples together with the speed of the product movement in the production line. This integration technique is expressed in One of the main issues regarding the active triangulation techniques described above for flatness measurement is how undesired movements of the product, such as bouncing, flapping and rotating, can affect the accuracy of on-line flatness measurements. These movements can be seen as vibrations of the product by the measurement system. In most modern flatness sensors based on active triangulation, two different techniques can be used to avoid introducing the effect of these vibrations in the flatness measurements. On the one hand, multiple arrays of spots or multiple stripes can be projected onto the product surface, as shown in An example of a flatness sensor using multiple arrays of laser spots to avoid the effects of undesired movements of the rolled product is an evolved version of the original Rometer. This sensor projects sets of two or three points in the longitudinal direction of the product surface. However, the approaches based on multiple light patterns impose several requirements. First, the patterns must be projected closely on the surface of the rolled product to ensure they suffer the same vibrations. Second, a highly accurate measurement of the speed of the product movement in the production line is required, since between two consecutive samples, some of the points acquired in the first sample under one pattern must be acquired in the second under another pattern. Therefore, the uncertainty in the measured speed introduces noise in the estimation of product vibrations and, thus, in the flatness measurements provided by the system. Furthermore, multi-plane camera focusing requires the lens of the machine vision system to operate with a smaller iris than when imaging only one plane. This is of utmost importance when working in harsh conditions with dark product surfaces or those covered with scale, where the patterns are reflected poorly and big apertures  in the lens are needed. Furthermore, camera calibration is more complex when several patterns are used. Finally, the cost of the systems increases exponentially, since a single-line pattern emitter usually costs more than the image sensor.As mentioned above, the second option to avoid the effect of undesired movements of the rolled products in flatness measurements consists of removing these movements from the surface reconstruction of the rolled products. Thus, this is a software approach that does not require extra hardware components. A more reliable surface reconstruction without vibration effects can be obtained using geometric transformations when the shape of the product is known previously . Further3.2.2.Fringe projection is an optical technique, where a set of parallel lines, or rectilinear fringes, are projected onto the surface of a product, as shown in Another optical technique that can be used to retrieve the shape of an object is moiré topography ,47. ThisrefL, is shorter than the length of the pattern projected onto the product surface.Vibration effects are not introduced in flatness measurements computed by systems using fringe patterns and moiré topography, since they sample the whole area of the product surface required to compute the flatness measurements simultaneously. That is, the reference length used to compute flatness measurements, 3.2.3.Recently, an optical technique based on diffused light was being used in the steelmaking industry for flatness measurement. This technique requires a very uniform, diffused light ribbon projected onto the surface of the product to be measured. The light ribbon is imaged by a CCD-matrix camera, as shown in Flatness sensors based on diffuse light projection can be used both in cold and in hot-rolling applications. Furthermore, since height movements do not affect the reflection angles of the light pattern, vibrations of the product do not add noise to the flatness measurements. This technique can only measure manifest flatness defects, similar to the rest of the optical techniques.3.3.An alternative, non-contact flatness sensor, SIFLAT, was developed by Siemens in 2005 . This flAlthough the principle used by this technique is contactless, systems implementing it require deflection rollers to ensure that no more vertical movement other than that exerted by the system appear in the product. Thus, in the system as a whole, some contact with the product surface is required. Therefore, this technique may not be suitable, as happens with other contact flatness sensors, for high quality surface manufacturing.3.4.Another non-contact technique used to measure the flatness of rolled products in the steelmaking industry is based on the capacitive effect. Distance measurements are retrieved taking the electrical features of the rolled product into account and are used to estimate the length of its fibers ,52. Flat4.Spain has traditionally been an important steel supplier. Several steel production plants were built in the 20th century, taking advantage of the mining resources of the country and the main maritime ports that allowed importing raw minerals from other countries. In the last decades of the century, a great effort was made to automate the industrial processes and measurement tasks of these plants. Usually, this automation—in continuous evolution—is carried out using both external and internal resources. Steel production plants usually rely on commercial systems and devices to automate process manufacturing and control or quality inspection. On other occasions, steel production plants use solutions specifically designed in their own research and development centers. In the second scenario, given the research orientation of the tasks, many times, these centers become a research partner with some universities.To the best of our knowledge, no Spanish company has designed or developed commercial flatness sensors for the steelmaking industry. In Spain, one example of a successful partnership between a steel technological center and a university is that between ArcelorMittal's R&D Technological Center, in Avilés, and the University of Oviedo, both in the Principality of Asturias. ArcelorMittal is the world's largest steel producer, and its plant in Asturias is the most important in the country. Several research projects have been collaboratively developed over the years, many of them in the field of measurement automation. Some of these projects resulted in an industrial prototype or a fully-functional system installed in one or more production lines of the plant. When proven successful, these systems are then exported to other plants of the steel producer. The need for ever-better flatness sensors in flat-rolling mills and production lines is a demand of this steel supplier —as it isAmong all the research groups at the University of Oviedo involved in this partnership, two of them are currently carrying out research projects on the automation of flatness measurement of flat-rolled products, both in cold and hot-rolling mills. The first group is with the Department of Computer Science and Engineering. This group has been working in the research, design and development processes of optical flatness sensors based on active optical triangulation for the steelmaking industry since the early 1990s. First, laser triangulation based on discrete points was studied, and a flatness sensor for hot-rolling mills was designed and developed using three laser spots projected onto the surface of the rolled products to be inspected –56. The The second flatness sensor designed by this research group was also based on spot-laser triangulation. In this case, five laser spots were projected onto the surface of the product under inspection, and stepping motors were used to both continuously track the longitudinal fiber of the surface and adapt to the width of each rolled product –59. The The drastic reduction in the costs of laser line projectors led to research tasks on reconstructing the whole surface of rolled products based on laser-stripe triangulation. Using surface reconstruction, metric measurements—like the length of longitudinal fibers—were taken, and flatness measurement was computed. A new flatness sensor was designed and developed by this research group ,60,61, fAs described above, one of the main drawbacks of flatness sensors based on a single laser stripe is the potential errors introduced in the flatness measurements, due to inaccurate surface reconstructions when undesired movements, such as vibrations, appear in the rolled product while manufacturing. Vibrations corrupt the height measurements, causing an erroneous reconstruction of the surface of the rolled product. Thus, flatness measurements are distorted, because the computed lengths of the longitudinal fibers with vibrations differ from the real lengths. As mentioned above, two different solutions can be applied to overcome this issue when using optical laser triangulation. First, multiple lines can be projected and used to accurately reconstruct the surface of the rolled product. Second, the effects of vibrations can be removed from the surface reconstruction before flatness measurements are taken. In the first case, the optical hardware is more expensive, since two or three laser stripes must be generated. This research group developed a new flatness sensor, where the effects of the vibrations introduced in the surface reconstruction of the rolled product are removed by software . In thisi) on-line and off-line visualization techniques to adequately provide flatness measurements to human operators in rolling mills [ii) compensation for uneven temperature across the width of the rolled product while rolling to avoid flatness defects caused by different temperature profiles [iii) modeling for flatness optimization in hot-rolling mill processes [Apart from the research in optical triangulation for automated flatness sensors in rolling mills, this research group developed, in collaboration with the aforementioned technological center, complementary research tasks for automated flatness measurement. Among them, the most important are:  coprofiles ; and . This group is focused on the application of the capacitive effect for distance measurement and has designed and developed a low-cost flatness sensor for cold-rolling based on this effect ,52.capacitor feeds a precision rectifier and a two-stage filter and amplifier that provide a DC signal as a function of the distance between the electrode and the rolled product. A finite element analysis is used to compute the relation between the capacitance and the distance. Finally, taking the distance measurements and the speed of the product movement in the manufacturing line into account, the length of the fibers is computed. The length of each fiber is computed using j, to the sensor electrode in the i-th sample. This flatness sensor has 24 electrodes of 52 mm each, with a nominal distance from the product surface of 5 cm. In this scenario, the capacitance to be measured is as low as 1 pF. Since this flatness sensor measures the length of the fibers using a non-contact technique, tensile stress across the width of the rolled product is not taken into account, and thus, only manifest flatness defects can be measured. Furthermore, since the sensor needs to be located near the rolled product, it is suitable neither for hot-rolling processes nor for areas in the processing lines where the movement of the product provokes severe undulations.This research group studied the use of capacitive distance measurement based on the electrical features of steel rolled products to retrieve the elongation of several longitudinal fibers of their surface. The distance measurement technique is implemented based on the classical impedance divider circuit, mainly intended to measure floating capacitors. In this design, a capacitor is built up using an electrode and the rolled product itself—the rolled product is considered the electrical ground. The output of this Another research group involved in this partnership that has developed research tasks in the field of flatness measurement automation is also with the EECS Department. This research group focused its research on measuring the straightness of rolled products in hot-rolling installations to quantitatively evaluate straightness deviations or curvature in the edges of the rolled products, commonly called camber . This reTo the best of our knowledge the above are the Spanish research groups working—or that have worked in the past—on flatness measurement automation for the steelmaking industry.5.State-of-the-Art Sensors Technology in Spain 2013, this paper also reviews the most important flatness prototypes and systems designed and developed for the steelmaking industry in Spain.In this paper, the main technologies and applications for on-line flatness measurement in the steelmaking industry have been reviewed. Most of them can also be used in other industries involving rolling mills or continuous production lines, such as aluminum, copper and paper, to name a few. Encompassed in the special issue, One issue concerning the design of modern flatness sensors is the accuracy of their measurements regardless of the harsh conditions of the rolling mill or the production line where the flat-rolled product is being manufactured. Mechanical-based flatness sensors provide accurate measurements though the tension of the rolled product, while manufacturing is not constant. On the other hand, optical-based sensors implement different approaches to remove the effects of undesired movements of the rolled product while manufacturing. These approaches can be hardware, projecting multiple patterns onto the product surface and using some invariants between these patterns to accurately compute the surface reconstruction of the product, or software, transforming and filtering the height measurements computed from a single pattern onto the surface to avoid noise in the surface reconstruction.Future flatness sensors for the steelmaking industry will be focused on providing real-time, high-density flatness maps, with more accurate measurements and very low uncertainty—modern on-line flatness sensors provide measurements with a relative uncertainty of 0.1% —in very


Recent studies have shown genetic and epigenetic aberrations resulting in aberrant activation of the Wingless-Int (Wnt) pathway, thus influencing the initiation and progression of acute myeloid leukemia (AML). Of major importance, these findings may lead to novel treatment strategies exploiting targeted modulation of Wnt signaling. This paper comprises the latest status of knowledge concerning the role of Wnt pathway alteration in AML and outlines future lines of research and their clinical perspectives. Acute myeloid leukemia (AML), a hematologic malignancy of the myeloid line of white blood cells in the bone marrow, is the most common leukemia of the adult , 2. DespDue to high mortality rates and high relapse rates even among transplanted patients, new therapeutic strategies are required , 5.The Wingless-Int (Wnt)/beta-catenin pathway has been shown to play an essential role in the regulation of cell proliferation, differentiation, and apoptosis of haematopoietic stem cells , 2, 4, 5Wnt (wingless) proteins constitute a family of cysteine-rich glycosylated proteins that contribute to lymphopoiesis and early stages of both B-cell and T-cell development –10. TheyTo date, 19 Wnt members have been identified in humans and more than eight mammalian Frizzled genes are known –13. Wnt Binding of Wnt either to Frizzled and the low-density lipoprotein receptor-related proteins (LRPs) 5 and 6 or to Frizzled protein alone results in the stabilization of beta-catenin, the major mediator of canonical Wnt signaling , 14. FriThree members of Dvl, Dvl-1, Dvl-2, and Dvl-3, have been characterized . They laα, δ, or ε) phosphorylates beta-catenin on serine 45. This step is independent of (GSK-3beta) and initiates the phosphorylation-degradation cascade of beta-catenin. In the following step, beta-catenin is phosphorylated at serine 33/37 by GSK-3beta [In unstimulated cells, axin in its phosphorylated form is able to bind beta-catenin effectively. Upon Wnt signaling, however, axin is dephosphorylated by the protein phosphatase 2A (PP2A). The dephosphorylated form of axin binds beta-catenin less efficiently than it does the phosphorylated form, thereby promoting the release of beta-catenin . A complSK-3beta . The phosphorylated beta-catenin is, subsequently, recognized by the E3 ubiquitin ligase subunit beta-transducin repeat-containing protein (beta-TrCP) and thereby targeted for ubiquitination and subsequent degradation by the proteasome , 18, 19.In the presence of Wnt signaling, the phosphorylating activity of GSK-3beta is inhibited leading to the stabilization of beta-catenin . This isc-myc, cyclin D1, and fibronectin [Dvl not only stabilizes beta-catenin in the cytoplasm but is also required in the nucleus where it interacts between c-Jun and beta-catenin, respectively. This mediates the formation of a Dvl-c-Jun-beta-catenin-TCF transcriptional complex, binding on the promoter of Wnt target genes . For exaronectin , 14, 19.Beta-catenin is a 92 kDa protein consisting of several structural domains , 21–23. in vivo conditions [Beta-catenin acts as a structural protein at cell-cell adherens junctions where it links cadherins to the actin cytoskeleton –34. In anditions , 38. Furnditions , 16, 39.nditions , 40, 41.The Wnt/beta-catenin pathway has been shown to play a critical role in the regulation of cell proliferation, differentiation, and apoptosis , 4–6 of in vivo still need to be clarified [It was recently published that AMLs are associated with an aberrant activation of the Wnt pathway, thus representing an attractive way of targeted therapeutic intervention . Howeverlarified , 6.in vitro without affecting cell viability in contrast to what is known for normal human CD34+ progenitor cells [Within the canonical Wnt signaling pathway, beta-catenin acts as the major effector molecule affecting self-renewal of hematopoietic stem cells while its dysregulation has been suggested in leukemic stem cell lines , 6–8. Abor cells , 10. It or cells , 8, 10. or cells , 7.Overexpression of beta-catenin seems to be an independent adverse prognostic factor, its overregulation could be found in the vast majority of AML samples , 4, 8. Iin vitro. Systemic application of these drugs in balb/c mice resulted in a decreased tumor growth and a prolonged overall survival [We recently identified several drugs as Wnt inhibitors , 42, 43.All these data greatly contribute to our understanding of the role of beta-catenin and the Wnt pathway in the development of AML. The canonical Wnt pathway could be shown to be of major importance in the pathogenesis of acute leukaemia. In spite of divergent results in recent studies influencing this pathway seems to be a promising treatment strategy and should be followed up in further studies for future clinical use in patients with AML.


Irradiation from diverse sources is ubiquitous and closely associated with human activities. Radiation therapy (RT), an important component of multiple radiation origins, is a common therapeutic modality for cancer. More importantly, RT provides significant contribution to oncotherapy by killing tumor cells. However, during the course of therapy, irradiation of normal tissues can result in a wide range of side effects, including self-limited acute toxicities, mild chronic symptoms, or severe organ dysfunction. Although numerous promising radioprotective agents have emerged, only a few have successfully entered the market because of various limitations. At present, the widely accepted hypothesis for protection against radiation-caused injury involves the Wnt canonical pathway. Activating the Wnt/β-catenin signaling pathway may protect the salivary gland, oral mucosa, and gastrointestinal epithelium from radiation damage. The underlying mechanisms include inhibiting apoptosis and preserving normal tissue functions. However, aberrant Wnt signaling underlies a wide range of pathologies in humans, and its various components contribute to cancer. Moreover, studies have suggested that Wnt/β-catenin signaling may lead to radioresistance of cancer stem cell. These facts markedly complicate any definition of the exact function of the Wnt pathway. Irradiation from diverse sources is ubiquitous and is closely associated with human activities. Among all sources of radiation  remains to be the only drug recommended as an effective radioprotective agent against xerostomia resulting from irradiation-in vivo researchet al.The Wnt signal transduction pathway (shorten as the Wnt pathway hereafter) is an evolutionarily conserved signaling pathway with an important role in fetal development and central nervous system formation, as well as in cell growth, migration, and differentiationThese pieces of evidence indicate that the Wnt/β-catenin pathway has a significant role in radioprotection. However, the deregulation of the Wnt/β-catenin pathway has been proposed as one of the signaling pathways responsible for carcinogenesis in the hematopoietic systems, the intestines, and the epidermis, as well as for chronic myelogenous leukemia, lung cancer, colorectal cancer and gastrointestinal cancer (GI)in vivo turnover in conduit blood vessels is approximately one to several years, with only 0.1% of cells actively proliferating in quiescent vesselsTissues are well organized and composed of cells with epithelial and connective tissue origins. Tissue regeneration ability varies with the cell source. First, continuously regenerating cells consist of the epithelial and hematopoietic bone marrow cells. Researchers have shown that the doubling time for regenerating clonogenic cells in the crypts of the small intestine is less than one day, and that the entire intestinal tract epithelial compartment renews completely within a few days,Given such distinct cellular compartmentalization, understanding the events occurring during and shortly after irradiation of tissues and cell is essential. This information is necessary in understanding the mechanisms of radioprotectors and mitigators. Death pathway modulators, which regulate early radiation damage, are among such radioprotective policies. This policy involves the activation of the Wnt/β-catenin pathways.The Wnt signaling pathway was discovered during an embryogenesis experiment on Xenopus laevis. The Wnt1 gene, the first gene of the Wnt protein family, was identified in 1982 as a gene activated by the integration of mouse mammary tumor virus proviral DNA into virally induced breast tumorsWnt ligands are a family of secreted glycoproteins involved in cell-to-cell communication that controls each major develop mental process, including cell-fate determination as well as cell proliferation, polarity, adhesion, motility, and apoptosis, and subsequently, patterning and morphogenesis,Cytoplasmic β-catenin is degraded by the “destruction complex” in the absence of a Wnt ligand binding to its receptor complex. In the destruction complex, Axin acts as a scaffold protein, in which adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK-3β), and casein kinase 1α (CK1α) bind to facilitate the sequential phophorylation of β-catenin in 45 serine by kinase CK1α and 41’threomine, and in 37’,33’serine by GSK-3β,,Dictyostelium discoideum and Xenopus laevisWith the increasing interest of researchers in the Wnt/β-catenin pathway, Wnt signaling is revealed to be modulated by numerous evolutionarily conserved inhibitors and activators. The Wnt/β-catenin pathway can be specifically blocked by endogenously secreted inhibitors from the Dickkopf (DKK) family, which interact with LRP5/6 and high-affinity receptors from the Kremen family, thus causing rapid endocytosis of Kremen-Dkk-LRP complex and removal of LRP from the cell membranesHead and neck cancer (HNC) is the fifth most common cancer, with an estimated annual incidence exceeding 500,000 worldwideet al.in vivo study that investigated the increase of the Wnt-1 expression in irradiated but viable acinar structures along with the membranous upregulation of β-catenin. In accordance with previous in vitro studies, Wnt pathway activation was demonstrated to provide a key radioprotective mechanism in irradiated cancer cells-To our knowledge, Hakim et al.To further understand the involvement of Wnt signaling in normal SG development and regeneration after injury. Hai et al.To further explore the potential of activating Wnt/β-catenin signaling in preventing radiation damage to salivary glands, Hai ,Oral mucositis, which is characterized by mucosal damage and inflammation in the oral cavity, is a common complication of chemotherapy and radiotherapy among cancer patients with head and neck tumors and hematological malignancies,in vitro and in vivo studieset al.The R-spondin family of proteins comprises novel secreted proteins acting as major agonists and modulators of the Wnt-β-catenin signaling pathwayet al.The LRP5/6 receptor also appears to play a role in activating the Wnt/β-catenin pathway. Wei In summary, Rspo1 demonstrates a protective effect against radioactive oral mucosa inflammation through the activation of the Wnt/β-catenin pathway. As a receptor protein for the Wnt signaling pathways, the internalization of LRP6 is an essential step in β-catenin activationNormal homeostasis of the intestinal epithelium is a dynamic balance maintained by an intricate cell replacement process in which terminally differentiated epithelial cells are continuously and rapidly replaced by replication and differentiation of epithelial cells (transit cells) located within the intestinal crypt. RIGS results from a combination of direct cytocidal effect on the intestinal crypt and on endothelial cells, and the subsequent loss of the mucosal barrier. This situation results in electrolyte imbalance, diarrhea, weight loss, infection, and mortality. DNA damage and, presumably, endoplasmic reticulum and mitochondrion damage induced by radiation-free radicals immediately trigger the pro-apoptotic cell responseet al.The β-catenin/TCF signal transduction pathway most probably has a significant role in regulating the proliferation and differentiation of intestinal epithelial cells during the regeneratiation and maturation processes along the crypt-villus axisGSK-3 located upstream of Wnt signaling, is involved in numerous important biological signal paths, and has an essential role in the Wnt/β-catenin pathway. In addition, GSK-3β, a type of GSK-3, is a major component of Wnt signaling which has an important role in the development and renewal of the intestinal epithelium by maintaining stem/progenitor cells and controlling migration and localization of epithelial cells along the crypt-villus axiset al.Thotala ,,The Wnt signaling pathway controls myriad biological phenomena throughout the development and adult life of all animals. In parallel, aberrant Wnt signaling underlies an extensive range of pathologies in humans, with its various components contributing to cancer development. Loss of APC function resulting from sporadic colorectal cancer leads to inappropriate stabilization of β-catenin and the formation of constitutive complexes between β-catenin and the intestinal TCF family member TCF4Genes mutated in at least 10% of the analysed samples for each cancer type are included in the table: APC, CTNNB1β-catenin, and WTX.et al.et al.Recently, several studies have investigated the possibility of using the Wnt/β-catenin signaling pathway for overcoming radiation resistance. Che et al.et al.Radiation is widely believed to affect the homeostasis of reactive oxygen species (ROS)To our knowledge, the Wnt/β-catenin pathway is an important signaling mechanism for cell proliferation. β-Catenin migrates to the nucleus after the inactivation of GSK-3β, which is followed by the gene expression of cyclin D1The Wnt/β-catenin pathway evidently has an important role in preventing damage induced by RT. The activation of the Wnt/β-catenin pathway, which provides a key radioprotective mechanism in irradiated cancer cells, exhibits protective effect against RT-induced oral mucositis and xerostomia. Moreover, activating Wnt/β-catenin signaling is a potential strategy for treating GI radiation syndrome. As a common signal pathway in the development and adult life of all animals, the Wnt signal transduction cascade, particularly the canonical Wnt pathway, is expected to occupy an indispensable position in treating normal tissue injuries, resulting from radiation exposure. Wnt/β-catenin signaling is associated with the development, renewal, and regeneration of numerous endoderm-derived organs; and research on this field has been ongoing for almost 40 years. Although a number of positive outcomes regarding the activation of the Wnt/β-catenin signaling pathway of antiradiation have been obtained, studies on the activation of the Wnt/β-catenin pathway related to antiradiation research remain extremely few to date. No drug based on Wnt/β-catenin signaling has been approved for radioprotective use in the clinical setting. The canonical Wnt pathway has been linked to cell proliferation in a variety of tissues and systems. Rspo1 is the only protein that can increase the mucosal thickness and reduce ulceration in the oral mucosa after irradiation and chemotherapy, presumably by increasing the proliferation of the mucosal epithelium in the basal layer of the tongue through the activation of the Wnt/β-catenin signaling pathway. The inhibitor of GSK3β SB216763 or SB415286 is studied only as a radioprotector primarily in animals because of its inclination to induce untoward effects in humans. The scientific community should further elucidate the side effects of the Wnt/β-catenin pathway to provide a potential therapeutic approach for preventing radiation-induced damage.From the preceding discussion, we firmly believe that the Wnt/β-catenin signaling pathway activator should be developed as a potential therapeutic approach to prevent radiation-induced damage in the future. At present, confirming such protective effects is essential, particularly in the clinical setting . However, considering the critical role of Wnt signaling in various cancer developments, the issue on specifically targeting the side effects of Wnt pathway-based drugs without increasing cancer risk should be addressed urgently.


Deregulation of Wnt/β-catenin signaling is a hallmark of the majority of sporadic forms of colorectal cancer and results in increased stability of the protein β-catenin. β-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto-oncogenes MYC and CCND1 as well as the genes encoding the basic helix-loop-helix (bHLH) proteins ASCL2 and ITF-2B. To identify genes commonly regulated by β-catenin in colorectal cancer cell lines, we analyzed β-catenin target gene expression in two non-isogenic cell lines, DLD1 and SW480, using DNA microarrays and compared these genes to β-catenin target genes published in the PubMed database and DNA microarray data presented in the Gene Expression Omnibus (GEO) database.Treatment of DLD1 and SW480 cells with β-catenin siRNA resulted in differential expression of 1501 and 2389 genes, respectively. 335 of these genes were regulated in the same direction in both cell lines. Comparison of these data with published β-catenin target genes for the colon carcinoma cell line LS174T revealed 193 genes that are regulated similarly in all three cell lines. The overlapping gene set includes confirmed β-catenin target genes like AXIN2, MYC, and ASCL2. We also identified 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are regulated similarly in DLD1 and SW480 cells and one pathway – the steroid biosynthesis pathway – was regulated in all three cell lines.Based on the large number of potential β-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T cells as well as the large overlap with confirmed β-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study Wnt/β-catenin signaling and associated colorectal carcinogenesis. Furthermore, the confirmed and the newly identified potential β-catenin target genes are useful starting points for further studies. The majority of sporadic forms of colorectal cancer are characterized by deregulation of Wnt/β-catenin signaling resulting in increased transcriptional activity of the protein β-catenin. In colon cells, a protein complex consisting of the protein Adenomatous polyposis coli (APC), the scaffolding protein Axin, casein kinase 1 (CK-1) and glycogen synthase kinase 3β (GSK-3β) tightly controls the transcriptional activity of the β-catenin protein by targeting it for proteasomal degradation. Stimulation of colon cells with Wnt proteins results in the disruption of the APC protein complex and stabilization of the β-catenin protein. β-catenin is then transported into the nucleus where it recruits cofactors of the TCF/LEF family to activate the transcription of its target genes ,3. Conseβ-catenin target genes have been implicated in regulating different cellular processes including proliferation , stem cell fate (ASCL2), survival , differentiation , migration MMP7, MMP14), and angiogenesis (VEGF) . While t, MMP14, Genes encoding members of the basic helix-loop-helix (bHLH) family of proteins, like ASCL2, HATH1, and ITF2, represent one of the largest groups of genes on the list of previously published β-catenin target genes ,19,20. MThe colon carcinoma cell lines, DLD1 and SW480, are frequently used as model systems to study colorectal carcinogenesis in general and β-catenin function in particular. To identify target genes that are commonly regulated by β-catenin, we chose these non-isogenic colorectal cancer cell lines for our experiments. While DLD1 cells are derived from a microsatellite instable (MSI) tumor, SW480 tumor cells are characterized by chromosomal instability (CIN) . In bothHere, we analyzed the gene expression profiles of DLD1 and SW480 cells after down regulation of β-catenin expression and compared the potential β-catenin target genes that are similarly regulated in DLD1 and SW480 cells with a comparable data set published for LS174T cells in the Gene Expression Omnibus (GEO) database as well as confirmed β-catenin target genes found in the PubMed database. We also identified 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are regulated similarly in DLD1 and SW480 cells. In addition, one KEGG pathway was regulated in all three cell lines. Based on the large number of genes that are regulated in a similar way in DLD1, SW480, and LS174T cells as well as the large overlap with previously published β-catenin target genes, we conclude that DLD1 and SW480 are good model systems to study β-catenin target genes and signaling pathways. Furthermore, we were able to confirm a large number of previously published β-catenin target genes and identified many new potential β-catenin target genes. Therefore, this data is a useful resource for further studies to elucidate the role of β-catenin signaling in colorectal cancer.To test the efficacy of the β-catenin siRNA used for our DNA microarray experiments, DLD1 and SW480 cells were treated with this β-catenin siRNA and β-catenin protein expression was analyzed using immuno detection. Since we observed the strongest down regulation of β-catenin protein after siRNA treatment for 48 hrs, cells were treated for this period of time. Compared to DLD1 and SW480 cells treated with control siRNA , treatment with β-catenin siRNA (siβcat) attenuated β-catenin protein expression levels in both cell lines  have previously been described as β-catenin target genes  38 out of these 66 genes are directly regulated by β-catenin as demonstrated by ChIP, EMSA, or RGA with mutated  TCF4 binding sites Table . When weDue to the large number of genes encoding members of the basic helix-loop-helix (bHLH) family of proteins among the list of β-catenin target genes  plugin, we searched the list of 193 genes that are differentially regulated in DLD1, SW480, and LS174T cells for known interactions. We identified three networks that contained three or more nodes (genes)  software ,81 to idNA Table . Apart fNA Table .and DLD1 cells (786 genes) as well as in LS174T and SW480 cells (677 genes), the number of commonly regulated genes in DLD1 and SW480 (335 genes) was low, resulting in a low number of commonly regulated genes in DLD1, SW480, and LS174T cells (193 genes). While DLD1 and SW480 cells both express a mutated form of APC, they differ with respect to the underlying genetic disturbance: DLD1 cells show microsatellite instability (MSI), whereas SW480 cells are derived from a tumor with chromosomal instability (CIN). However, SW480 cells (CIN) share a lot of commonly regulated genes with LS174T cells (MSI), suggesting that other factors apart from genetic instability contribute to the observed differences in gene expression between DLD1 and SW480 cells. Looking at the mutational status, DLD1, SW480, and LS174T cells differ with respect to the status of the genes APC, PI3K, and TP53, but they are identical with respect to status of the genes KRAS and PTEN: DLD1 , SW480 , and LS174T cells  [β-catenin plays a crucial role in embryogenesis, tissue homeostasis and carcinogenesis. Because of its role in regulating homeostasis of the intestinal tract as well as being a key player in sporadic forms of colorectal cancer, inhibition of β-catenin is not a suitable strategy to treat patients with colorectal cancer. For this reason, identifying and characterizing β-catenin target genes could result in better understanding of colorectal carcinogenesis and development of new therapies. Here, we used two non-isogenic colorectal cancer cell lines, DLD1, and SW480, and identified 335 commonly regulated β-catenin target genes. 193 of these genes were also differentially regulated in LS174T cells.  Compared to the number of differentially regulated genes in LS174T TEN: WT) ,83.ApcMin/+ mice resulted in an increased tumor number and size and implicated the CCND1 gene as one transcriptional target that contributes to the observed phenotype of these mice [Deregulation of PI3K/Akt signaling (as observed in DLD1 and LS174T) has been implicated in the phosphorylation of β-catenin at Ser552 , therebyese mice . In our ApcMin/+ mice: Despite the fact that p53 protein expression is deregulated as a consequence of APC loss (and probably other factors) and p53 is known to regulate β-catenin expression via a negative feedback loop, loss of p53 did not promote the initial steps of intestinal neoplasia in ApcMin/+ mice [The tumor suppressor p53 has been implicated in down-regulating β-catenin expression and/or activity by Siah-1 dependent (and GSK-3β independent) degradation ,89, redun/+ mice , suggestApcmNLS/mNLS mice with Apc+/+ mice, they found up regulation of the mRNA for the genes AXIN2, MYC, and CCDN1 as well as down regulation of HATH1 mRNA in response to stimulation with Wnt [Apart from regulating β-catenin protein expression levels via proteasomal degradation, (nuclear) APC has been implicated in affecting β-catenin activity by recruiting co-repressors of β-catenin to promoter regions, as well as sequestering and enhancing the nuclear export of β-catenin -96. Mutawith Wnt . AccordiThe differential expression of the genes AXIN2 and CCND1 in the three colorectal cancer cell lines highlights the fact that the genetic background of a given cell has a major impact on the expression of a specific gene. However, linking a specific somatic mutation in a tumor suppressor gene or proto oncogene, e.g. APC, PI3K, or TP53, to these differences in expression is difficult because the expression of a specific gene is affected by multiple transcription factors . Therefore, we are not able to attribute the observed differences in the gene expression profiles for DLD1 and SW480 to a single mutation.and SW480 or DLD1, SW480, and LS174T cells, assuming that these genes are commonly and directly regulated by β-catenin.Compared to LS174T cells, the total number of differentially regulated β-catenin target genes was much lower in DLD1 and SW480 cells. This could be either specific to the genetic background of the cell lines used or due to different experimental approaches. While we transiently transfected DLD1 and SW480 cells with β-catenin siRNA and analyzed the gene expression 48 hours after transfection, Mokry et al.  stably tRemarkably, the number of β-catenin regulated genes was very similar in the three cell lines when focusing on the list of 66 previously described β-catenin target genes. Here, DLD1, SW480 and LS174T cells differentially regulated 21, 30 and 34 of these genes, respectively. There are at least two reasons why the number of detected β-catenin target genes did not include  all 66 β-catenin target genes presented in Table and SW480 cells that are differentially regulated in response to treatment of the colorectal cancer cell lines with β-catenin siRNA. In addition, the steroid hormone biosynthesis pathway was similarly regulated in all three cell lines analyzed. Whereas the steroid hormone biosynthesis pathway [2+ signaling pathway under one database entry, despite different functions of these signaling pathways. While the canonical Wnt pathway has been implicated in regulating the activity of the β-catenin protein, PCP signaling results in remodeling of the cytoskeleton that is a prerequisite for migration and cell polarization via the GTPases RHOA and RAC1. Wnt/Ca2+ signaling, however, plays a role in the regulation of cell adhesion and cell movements during gastrulation and results in the activation of protein kinase (PKC), calcium calmodulin mediated kinase II (CAMKII) and calcineurin [Using the KEGG pathway database, we identified 11 signaling pathways or cellular functions in DLD1  pathway ,102 as w pathway ,104, the pathway ,106, apo pathway ,108, reg pathway , and foc pathway , have becineurin .and SW480 cells have been associated with Wnt/β-catenin signaling based on PubMed publications: the m-calpain pathway [Furthermore, all three Biocarta pathways that were enriched in DLD1  pathway ,112, the pathway ,113, and pathway ,115.Since 6 out of the 66 previously described β-catenin target genes encode proteins that belong to the bHLH protein family Table , we focuIn conclusion, by comparing potential β-catenin target genes in DLD1 and SW480 cells with 1) corresponding data from LS174T cells and 2) a list of previously identified β-catenin target genes, we found and confirmed a large number of β-catenin target genes, suggesting that DLD1 and SW480 (as well as LS174T cells) are suitable for identifying β-catenin target genes as well as β-catenin dependent signaling pathways and functions. Therefore, the presented list of commonly regulated genes in DLD1 and SW480 cells as well as the annotated list of previously published β-catenin target genes are useful resources for further studies.The colon carcinoma cell lines DLD1 and SW480 were obtained from ATCC  and were cultured in DMEM medium supplemented with 10% calf serum . Short interfering RNAs targeting β-catenin (cat. no. D-003482-04) and β-galactosidase (cat. no. D-012539-01) were purchased from Dharmacon  as siGenome siRNAs. Cells were transfected with these siRNAs using Lipofectamine 2000 .Cell lysates were prepared with reporter gene lysis buffer  supplemented with protease inhibitor cocktail I . Equal amounts of protein were mixed with 2× SDS loading buffer  glycerol; 10% (w/v) SDS; 100 mM Dithiothreitol; 0.01% (w/v) bromphenol blue), separated by electrophoresis in discontinuous SDS-polyacrylamide gels and transferred to Immobilon-P membranes . Antibodies specific for β-catenin , β-actin  and the secondary horseradish peroxidase-conjugated goat anti-mouse antibody  were used for immuno detection. Blots were subjected to Supersignal West Dura chemiluminescence substrate  and exposed to Fuji Medical x-ray film SuperRX (FujiFilm).5 DLD1 or SW480 cells were seeded per well (12 well plate). Cells were transfected with the indicated siRNAs using Lipofectamine 2000 (Life Technologies). 24 hours later, the 8×TOPflash reporter gene construct  as well as the plasmid pCH110 encoding β-galactosidase  were transfected into these cells. 72 hours after the siRNA transfection, cells were harvested, lysed with reporter lysis buffer (Promega) and luciferase activities were measured using a luciferase assay reagent (Promega) and a luminometer . β-galactosidase activity was determined by standard methods using o-Nitrophenyl β-D-galactopyranoside  as a control for transfection efficiency.For reporter gene assays, 10DLD1 and SW480 cells were harvested 48 h after siRNA transfection using TRIzol . Total RNA was isolated according to the manufacturer’s instructions and reverse transcribed using SuperScript II reverse transcriptase (Life Technologies) according to the instructions provided by the manufacturer. cDNA was amplified and labeled with biotin using kits from Affymetrix. Labeled probes were hybridized to Human Genome U133 Plus 2.0 microarrays (Affymetrix), washed, stained and scanned using equipment from Affymetrix. The microarray analysis was performed in triplicates for each cell line .normal colon or colon carcinoma cells or colon carcinoma cell lines. For this reason, the PubMed database was searched using the phrase “beta catenin AND target AND ”. 591 articles were identified  and publications were screened for the identification and characterization of β-catenin target genes. We were able to identify 66 published β-catenin target genes that matched our criteria (Table http://www.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes (last update: October 2010). These genes are marked with an asterisk after the gene name (Table We were particularly interested in β-catenin target genes that are regulated in http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18560). The software package R (version 2.14.1) was used for the statistical analysis of the microarray data. Microarray data were preprocessed using the rma function from the R package “affy”. The expression matrix was filtered to contain only those probe sets that can be assigned to a gene name. Log2 intensity values were used for all subsequent analyses. Differentially expressed genes were identified with the R package “limma”. Genes were called “differentially expressed” if the corrected p value (according to Benjamini-Hochberg) was lower than 0.05. We considered a gene to be significant only if all of its significant probe sets were regulated in the same direction. Genes with significant probe sets that were differently regulated were not considered significant because we could not determine if the gene is up- or down regulated.For evaluation purposes, we compared genes differentially expressed in DLD1 and SW480 cells after treatment with β-catenin siRNA to a previously published data set containing differentially expressed genes after treatment of the colon carcinoma cell line LS174T with β-catenin shRNA  [KEGG and Biocarta pathways that are regulated similarly in two or three cell lines were identified using the Biovenn software (iovenn/) .Gene symbols of β-catenin target genes common to two or more cell lines were entered in the query function of the “Michigan molecular interaction” (MiMI) plugin (version 3.11) in the software package “Cytoscape” (version 2.83) ,120. Usihttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44097.The data sets supporting the results of this article are available NCBI's Gene Expression Omnibus (GEO) repository . The uniThe authors declare that they have no competing interests.AH analyzed the data, generated the figures and drafted the manuscript. VJ performed the statistical analysis. ST and SK performed the DNA microarray experiment. HB, BG and FTK participated in the design and coordination of the study. All authors read and approved the final manuscript.Results of the DNA microarray analysis . DLD1, SW480, and LS174T cells were treated with siRNA or shRNA targeting beta-catenin. Differentially expressed genes were identified using DNA microarrays as described in Materials and Methods. Using the Biovenn software, genes that are regulated similarly in two or three cell lines were identified and listed under separate tabs. Accordingly, genes that are differentially expressed in only one cell line were listed in a separate tab labeled with the name of the cell line.Click here for fileResults of the DNA microarray analysis (focusing on known β-catenin target genes). DLD1, SW480, and LS174T cells were treated with siRNA or shRNA targeting β-catenin. Microarray data was re-analyzed focusing our analysis only on the list of these 66 previously published β-catenin target genes. Using the Biovenn software, genes that are regulated similarly in two or three cell lines were identified and listed under separate tabs. Accordingly, genes that are differentially expressed in only one cell line were listed in a separate tab labeled with the name of the cell line.Click here for fileResults of the DNA microarray analysis (focusing on genes encoding bHLH proteins). DLD1, SW480, and LS174T cells were treated with siRNA or shRNA targeting beta-catenin. Microarray data was re-analyzed focusing our analysis only on the 96 bHLH proteins listed in the PFAM database. Using the Biovenn software, genes that are regulated similarly in two or three cell lines were identified and listed under separate tabs. Accordingly, genes that are differentially expressed in only one cell line were listed in a separate tab labeled with the name of the cell line.Click here for fileGSEA analysis using the Biocarta pathway database. This zipped file contains confirming data of the GSEA analysis. The names of the directories containing the files were composed of the term ‘GSEA’, the name of the cell line, e.g. DLD1, SW480, or LS174T, and the pathway database (Biocarta). Please use a web browser to view the files with the name ‘index.html’ in the corresponding directories to start exploring the data.Click here for fileGSEA analysis using the KEGG pathway database. This zipped file contains confirming data of the GSEA analysis. The names of the directories containing the files were composed of the term ‘GSEA’, the name of the cell line, e.g. DLD1, SW480, or LS174T, and the pathway database (KEGG). Please use a web browser to view the files with the name ‘index.html’ in the corresponding directories to start exploring the data.Click here for file


In the last decade there have been made experimental approaches targeting the Wnt pathway in chronic leukemia. In this paper we provide an overview about the current state of knowledge regarding the Wnt/β-catenin signaling pathway in chronic leukemia with special focus on therapeutic options and strategies.It has been revealed that the Wnt/ Chronic myeloid leukemia (CML) is a one of the myeloproliferative diseases and represents 15–20% of all adult leukemia types. Depending on clinical aspects and laboratory findings, CML can be divided in three phases. Typically CML begins in the chronic phase, and over several years it progresses to the accelerated phase and finally to the blast crisis. By the introduction of the bcr-abl tyrosine kinase inhibitor (TKI) imatinib and the second-generation TKIs dasatinib and nilotinib, the treatment of CML has been revolutionized. The overall survival rate of patients with CML has been clearly improved. Nevertheless, resistance and incompatibilities against TKIs are well known, and a molecular remission is difficult to achieve [Chronic lymphocytic leukemia (CLL) belonging to the indolent B-non-Hodgkin lymphoma (B-NHL) is the most common leukemia in the adult. The treatment of CLL mainly depends on the clinical stage of the disease, which is determined by the Binet staging system (A–C). In the early stage a “watch and wait” strategy is recommended. In progress of CLL, various chemotherapeutical options are determined. The application of fludarabine, cyclophosphamide, and rituximab (FCR) is one of the treatment standards in the first-line therapy of CLL . Furtherβ-catenin signaling pathway is focused. Influencing the expression of genes involved in cell proliferation, differentiation and apoptosis are primarily due to the activation of the canonical β-catenin and lymphoid enhancer factor (LEF)/T-cell factor (TCF) pathway [β-catenin that has two essential functions: it is a very important element of many intracellular signaling pathways, including the Wnt pathway, but it also takes part in creating intercellular adhesive junctions [β-catenin is mainly regulated by modulating its degradation and nuclear translocation [The Wingless-Int (Wnt) signaling pathway plays an essential role in embryogenesis and proliferation, survival, and differentiation of hematopoietic stem cells (HSCs) , 4. It r pathway . Wnt prolocation .α), and β-catenin form the destruction complex [β-catenin by GSK3 acting in conjunction with APC and axin causes β-catenin to interact with the ligase β-transducin repeat-containing protein (β-TrCP) which results in its ubiquitination and degradation, executed by proteasome [β-catenin is achieved in the nucleus. Here, the transcription factor of the LEF/TCF family, together with other proteins such as groucho, binds to DNA and inhibits gene expression as a transcriptional repressor , axin, glycogen synthase kinase 3 (GSK3), casein kinase alpha  family of proteins and their coreceptors lipoprotein receptor-related protein 5 and 6 (LRP 5 and 6) . Consequeveloped , 11. In  factors . In the nd c-Myc , 13 , casein kinase II (CK II), integrin-linked kinase (ILK), B-cell lymphoma 9 (BCL9) and BCL9-2/BCL9-like (B9L), together with pygopus [β-catenin pathway and lead to an increased accumulation of cellular β-catenin with augmented assembly of Wnt target genes. Axin1, axin2, HMG-box transcriptional factor (HBP1), Dapper1 (Dpr1), Chibby, and inhibitor of β-catenin and TCF (ICAT), on the contrary, represent complex negative regulators of the Wntsignaling pathway. They antagonize the Wnt pathway by decreasing the cellular β-catenin, disrupting the β-catenin/LEF-1 complexes, and repressing the Wnt target genes [There have been defined many other agents that work as physiological promoters of the Wnt pathway, such as dishevelled-associated kinase (DAK), casein kinase I epsilon  remain in the patient. In vivo after imatinib treatment, LSCs not only remained but also accumulated increasingly in bone marrow of CML mice . In thisuced CML . Intriguse cells . A compat aspect .β-catenin signaling may contribute to the defect in apoptosis that characterizes this malignancy [β-catenin due to Wnt stimulation, it cooperates with the transcription factor LEF-1, which is overly expressed in CLL by more than three-thousand-fold compared to normal B cells [β-catenin signaling components which fundamentally influence CLL cell survival [A significant role in the development of CLL is attributed to the Wnt pathway. It has been demonstrated that the Wnt signaling pathway is activated in CLL cells and that uncontrolled Wnt/lignancy . It is tlignancy . In primlignancy . After u B cells . Furthersurvival , 33. Simβ-catenin pathway. The balance between epigenetic downregulation of negative effector genes and increased expression of positive effector genes plays an outstanding role in the pathogenesis of CLL [It has been revealed that certain Wnt and Wnt network target genes are expressed at higher or lower levels in CLL compared with normal B cells . This ins of CLL . Especias of CLL , 34.Furthermore, the overexpression of positive effectors in the Wnt pathway is also involved in the pathogenesis of CLL. In this model, DNA methylation, histone modifications, and altered expression of microRNA molecules interact to allow continuous Wnt signaling. All these mechanisms result in a common consequence, the activation of LEF-1/TCF transcription factors and subsequent target gene expression .β-catenin-regulated transcription factor LEF-1 and its downstream target cyclin D1, are overexpressed in CLL. Lu et al. were able to demonstrate that a pharmacological inhibitor of GSK3β, SB-216763, activated β-catenin-mediated transcription and enhanced the survival of CLL lymphocytes [A large number of Wnt proteins such as Wnt3, Wnt5b, Wnt6, and Wnt10a, as well as the Wnt receptor Fzd3, are highly expressed in CLL, compared with normal B cells. Furthermore the Wnt/phocytes . Taken tphocytes .Dickkopf1 (DKK1) is known to antagonize Wnt signaling by direct high-affinity binding to the extracellular domain of the Wnt coreceptor LRP6. In B cells from patients with CLL, added DKK1 did not inactivate the Wnt pathway. The reason for this could be the absence of the binding domain of LRP6. It is estimated that in CLL cells every 6th LRP6 receptor is lacking the extracellular domain. Normal B cells proved to have significantly higher levels of extracellular, DKK1-binding domain of LRP6. On the other hand, a truncated LRP6 without extracellular DKK1-binding domain could lead to an uncontrollable activation of Wnt signaling in CLL .β-catenin signaling is important for the proliferation of CSCs. Therefore, inhibition of Wnt/β-catenin signaling is a promising treatment approach [β-catenin/LEF-1/TCF activation offers multiple options to target the aberrantly activated pathway in order to prevent target gene expression or their gene products to exert their tumorigenic function [Cancer stem cells (CSCs) play a significant role in the development and recurrence of several cancers. At the same time Wnt/approach . While sapproach . Howeverapproach . The varfunction .β-catenin pathway in tumor cells, from antagonizing Wnt ligand secretion or binding to promoting β-catenin degradation to specifically blocking β-catenin-mediated transcriptional activity [Over the last decade, numerous approaches have been developed to target the Wnt/activity . The binactivity . There hactivity , 39–41.β-catenin in the Wnt signaling pathway is essential for survival and self-renewal of LSCs in CML, providing a new strategy for targeting these cells [Little is known about targeting the Wnt pathway in CML. Based on current knowledge, there have not been made breakthrough advances targeting the Wnt pathway in CML. However, the fact remains that se cells . Particuβ-catenin signaling [In contrast, recent researches indicate that the Wnt pathway is an attractive candidate for developing targeted therapies for CLL . As one ignaling .β-catenin signaling. Cell viability assays showed that EA selectively induced cell death in primary CLL cells. Exposure of CLL cells to EA decreased the expression of Wnt/β-catenin target genes, including LEF-1, cyclin D1, and fibronectin. Immune coprecipitation experiments revealed that EA could directly bind to LEF-1 protein and destabilize the LEF-1/β-catenin complex [β-catenin signaling [Furthermore Lu et al. identified the diuretic agent ethacrynic acid (EA) as a Wnt inhibitor. In vitro assays confirmed the inhibitory effect of EA on Wnt/ complex . Similarignaling   has been shown to possess an antineoplastic effect in Wnt/i et al. . Apoptos therapy .β-catenin/LEF-1 pathway (CGP049090 and PKF115-584) in vitro and in vivo studies in order to antagonize LEF-1 [β-catenin/LEF-1 interaction. In vivo studies exhibited tumor inhibition of 69% with CGP049090 and 57% with PKF115-584 [Gandhirajan et al. used two small molecule inhibitors of the Wnt/ze LEF-1 . By usinze LEF-1 . The twoF115-584 . Targetiβ-catenin signaling pathway plays an outstanding role in the development of chronic leukemia. A large number of the aberrant regulation mechanisms in the Wnt pathway that lead to maintenance, progress, and relapse of chronic leukemia have been revealed. Furthermore, in the recent years, there have been made promising experimental, preclinical researches, targeting the Wnt pathway, particularly successfully in CLL. Further preclinical studies and first clinical results remain to be seen. The Wnt/


SSEE) but not wild-type IKKβ into monocytes stimulates differentiation of bona fide osteoclasts in the absence of RANK ligand (RANKL). This phenomenon is independent of upstream signals because IKKβSSEE induced the development of bone-resorbing osteoclasts from RANK and IKKα knockout monocytes and in conditions in which NEMO-IKKβ association was inhibited. NF-κB p100 and p105, but not RelB, were critical mediators of this effect. Inflammatory autocrine signaling by tumor necrosis factor α (TNF-α) and interleukin 1 (IL-1) were dispensable for the spontaneous osteoclastogenesis driven by IKKβSSEE. More important, adenoviral gene transfer of IKKβSSEE induced osteoclasts and osteolysis in calvariae and knees of mice. Our data establish the sufficiency of IKKβ activation for osteolysis and suggest that IKKβ hyperactivation may play a role in conditions of pathologic bone destruction refractory to RANK/RANKL proximal therapeutic interventions. © 2010 American Society for Bone and Mineral Research.Monocytes differentiate into osteoclasts through stimulation of receptor activator of NF-κB (RANK). Many downstream effectors of RANK play a positive role in osteoclastogenesis, but their relative importance in osteoclast differentiation is unclear. We report the discovery that activation of a single pathway downstream of RANK is sufficient for osteoclast differentiation. In this regard, introduction of constitutively activated IKKβ (IKKβ Bone balance depends on the concerted activity of osteoblasts, bone-forming cells, and osteoclasts, bone-resorbing cells. In pathologic conditions such as rheumatoid arthritis and osteoporosis, bone balance favors increased osteoclast activation, resultinβ3-integrin, cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metalloproteinase 9 (Mmp9).(The osteoclast differentiates from monocyte precursors through the action of ligand for the receptor activator for NF-κB (RANKL) and macrophage colony-stimulating factor (M-CSF). On stimu9 (Mmp9). With exp9 (Mmp9).–9 These 9 (Mmp9).The complement of proteins that are important for osteoclast differentiation makes up a growing list, but their relative importance remains unclear. A well-studied family of transcription factors that is required for osteoclastogenesis is NF-κB. NF-κB p100 and p105 are both required together for osteoclast differentiation. AdditionThe IκB kinase (IKK) complex activates NF-κB downstream of RANK. Upstream signals lead to association of two catalytically active kinases, IKKα and IKKβ, with the noncatalytic member IKKγ/NEMO. This association is required for activation of IKK through phosphorylation of two IKK activation-loop serines. IKK then phosphorylates IκB, targeting it for proteasomal degradation and allowing NF-κB to enter the nucleus and regulate gene transcription.–16 PharmSSEE), we reveal a signal for differentiation of functional osteoclasts that occurs downstream of, but independent from, RANK. IKKβSSEE, but not wild-type IKKβ or IKKαSSEE, induces osteoclast differentiation from monocytes. This phenomenon depends on NF-κB but does not require NEMO, IKKα, or RelB. Finally, adenoviral gene transfer of IKKβSSEE in knees and calvariae of mice is sufficient for development of massive osteolysis. Our findings demonstrate for the first time that a single activated kinase is sufficient for RANK-independent osteoclast differentiation and that active IKKβ induces osteolytic disease. These data highlight the centrality of IKKβ in osteoclast differentiation and implicate hyperactivation of IKKβ in pathologic bone destruction.We now report that IKKβ is not only necessary for RANKL-mediated osteoclastogenesis, but its activation also is sufficient for osteoclast formation. Using retroviral delivery of constitutively active IKKβ . -Cre mice were frooxed IKKβ mice wergous mice were obt knockout and cont knockout and cont-knockout and contIKKα was kindly provided by Dr Kenneth Marcu . IKKβ and RelB cDNA were purchased from ATCC . RelA cDNA was provided by Dr C Sasaki . All expression constructs were subcloned into pMxs using standard techniques. The following mutations were generated using the QuickChange II Site Directed Mutagenesis Kit  with primer pairs in parentheses: IKKβSSEE ; IKKβWWAA ; IKKβKM ; and IKKαSSEE . Note that the constitutive activating effect of the mutation of IKKβ was established previously.. Mouse cDNA for eviously.,292, at which point they were infected with retrovirus . Twenty-four hours after infection, cells were selected in α-MEM containing 10% FBS, 10 ng/mL of M-CSF, 100 U penicillin/100 µg strep per Liter, and 2 µg/mL puromycin for 72 hours, at which point selection medium was removed, and cells were washed and grown for 24 additional hours without puromycin. At this point, cells were lifted, counted, and plated for downstream experiments.Marrow was flushed from long bones into α minimum essential medium (α-MEM). Spleens and day 18.5 fetal livers were crushed into cell suspensions in α-MEM and were centrifuged at 453 rcf. Cell pellets were resuspended in whole medium . Monocytes/macrophages were produced by growing cell suspensions in the presence of 10 ng/mL of M-CSF. Monocytes/macrophages were allowed to proliferate for 3 days at 37°C in 5% COThe use of Plat-E retrovirus packaging cells stably expressing retroviral structural proteins gag-pol and env for transient production of high-titer retrovirus was described previously. Briefly,4 monocytes were plated in 200 µL of α-MEM with 10% FBS. IKKβSSEE-expressing cells were cultured in 10 ng/mL of M-CSF, whereas GFP- and IKKβWT-expressing cells were cultured in 10 ng/mL of M-CSF plus 100 ng/mL of RANKL for 4 days. At this point, cells were fixed and TRACP stained using the Leukocyte Acid Phosphatase Kit . TRACP+ cells with three or more nuclei were scored as osteoclasts.For osteoclastogenesis assays, 3 × 10OPG/Fc chimera , 25 µM TAT-NBD (YGRKKRRQRRR-G-TTLDWSWLQME) or 25 µM of TAT–mutant NBD (YGRKKRRQRRR-G-TTLDASALQME) during the entire course of retroviral transduction and in vitro osteoclast differentiation.For inhibition of osteoclastogenesis, cells were treated with 100 ng/mL of RNA was isolated from macrophage or osteoclast cultures using the Total RNA Isolation Mini Kit  according to manufacturer's instructions. Reverse transcription was described previously.The real-time quantitative PCR (qPCR) procedure was described in detail previously.The Western blot procedure was described previously. One millWT, flag IKKβWA, flag IKKβSSEE, or flag IKKβSSEE/WA were lysed in immunoprecipitation (IP) buffer  at 4°C. Protein was measured by BCA Assay  and normalized. Nonspecific binding was removed by rocking total cell lysate at 4°C with GammaBind G Sepharose beads  and 100 ng of normal mouse IgG for 2 hours at 4°C. Beads and normal antibody were centrifuged, and supernatant was incubated with GammaBind G Sepharose beads and 1 µg/mL of mouse anti-Flag M2 antibody (Sigma) in a total of 700 µL of IP buffer and 1× protease inhibitor cocktail at 4°C for 16 hours. Immune complexes were centrifuged with beads. Supernatant was removed by vacuum suction, and 2× sample buffer  was added to the beads, which were boiled for 5 minutes to elute the complex components, which were analyzed by Western blot.One million cells expressing GFP, flag IKKβ2, and 16 µM ATP. The reaction was terminated with 30 µL of reducing sample buffer. Samples were analyzed by Western blot.Plat-E cells expressing indicated flag-tagged IKKβ constructs were lysed in IP buffer. IKKβ was immunoprecipitated with M2 antibody, washed twice with IP buffer and once with kinase assay buffer , and incubated for 30 minutes at 30°C in 30 µL of kinase assay buffer with 1 µg GST-IκBα, 2.5 mM MgCl2 100-µm-thick dentin slices for 5 days in a 48-well tissue culture plate. To visualize resorption pits and tracks, slices were exposed to 0.5 N NaOH, and cells were removed by mechanical agitation. Slices were washed in PBS and stained with 0.1% toluidine blue (w/v) in PBS. Stained slices were rinsed with PBS and blotted dry, and pits were visualized by light microscopy. Resorption of artificial matrix was described previously. for 10 minutes in a dark, humidified chamber at room temperature. Slices were washed with PBS and mounted onto microscope slides for visualization of actin rings with fluorescent microscopy.IKKβSSEE was generated by subcloning from the pMx parental vector into Ad5 CMV K-NpA Shuttle using EcoR1 and Not1 restriction endonucleases . Recombination, and control (Ad ntLacZ). Five mice were used for these experiments with comparable results. For calvarial osteolysis, 1 × 107 pfus of virus diluted in 50 µL of sterile PBS were injected supracalvarially. Lipopolysaccharide (LPS) (10 µg) or RANKL (4 µg) diluted in 50 µL total volume of PBS were injected as positive controls. For calvarial experiments, five mice were used for each condition  with comparable results. In vivo injections were executed with 100-mL insulin syringes with 29G needles. Seven days after injection, knees and calvariae were fixed, decalcified, and analyzed histologically for osteoclasts and osteolysis.Adenovirus expressing bination, producti4NOH, pH 7.2], dehydrated in ethanol (30% to 70%), cleared through xylene, and embedded in paraffin. Sections were stained histochemically for TRACP to visualize osteoclasts or hematoxylin and eosine (H&E) to assess tissue architecture. Immunohistochemistry was performed according to the antibody manufacturer's instructions for immunoperoxidase staining.Bones were collected from mice and fixed in 10% buffered formalin for 24 hours. Bones then were decalcified for 7 days in buffer consisting of [14% (w/v) EDTA and HCells and histologic sections were imaged under white or ultraviolet (UV) light on an inverted microscope (Olympus IX-51). For f-actin visualization, UV light was passed through an FITC filter cube to localize green phalloidin. Digital images were captured using a CCD camera .t test for comparison between means was used for all analyses.Student's two-tailed IKKβ that could prevent or enhance its ability to rescue osteoclast differentiation in cells lacking IKKβ, we made the observation that constitutively activated IKKβ (IKKβSSEE), but not the wild-type (IKKβWT) form, in wild-type or IKKβ knockout bone marrow–derived macrophages induced the formation of osteoclasts in the absence of RANKL  and authentic bone matrix (SSEE by RANKL-independent osteoclasts was demonstrated using IKKβSSEE-GFP fusion construct (not shown). These data provide evidence that the TRACP+ multinucleated cells induced through expression of constitutively active IKKβ in macrophages are authentic osteoclasts.Further characterization showed that IKKβntiation.,32 IKKβSesorption,33 . Actin rings and resorption pits were observed in IKKβWT-infected cells only after RANKL administration (not shown). These observations reveal that IKKβSSEE is sufficient to induce an authentic program for functional osteoclasts from adult and fetal precursor cells independent of RANKL. To verify the specificity of the osteoclastogenic effect of the phosphomimmetic mutation, we mutated IKKβ activation-loop serines to alanine (IKKβSSAA) and lysine to methionine (IKKβKM), respectively. These mutations resulted in an activation-deficient molecule that failed to rescue RANKL-induced osteoclastogenesis in IKKβ knockout monocytes ,34 and Rut RANKL A. Furtheut cells B. Importto RANKL B. IKKβSSd dentin C. Consishepsin K D, indicanclusion E. TherefSSEE induces osteoclastogenesis independent of RANK, we tested whether IKKβSSEE also could induce osteoclastogenesis in the absence of NEMO binding. First, we determined that while administration of cell-permeable NBD peptides, which inhibit the association of IKKβ with NEMO, blocks RANKL-induced osteoclast differentiation, NBD did not inhibit osteoclastogenesis in response to transduction of IKKβSSEE (SSEE/WA) prevent IKKβSSEE from binding to NEMO , with NEMO.,36 SinceIKKβSSEE A. Second to NEMO B, C, IKKogenesis D. This qogenesis E, F. TheSSEE could induce osteoclastogenesis without the classical IKK complex, which includes IKKα, a kinase that is required for osteoclastogenesis in vitro. do not differentiate into osteoclasts  spleen macrophages with GFP, IKKβWT, and IKKβSSEE , which is usually considered to be a function of IKKα., we sought to determine whether these inflammatory factors were required for IKKβSSEE to induce osteoclast differentiation. Using IKKβSSEE-transduced TNF-α or IL-1 receptor knockout monocytes, we found that TNF-α and IL-1 are not required for IKKβSSEE to accomplish its effect in osteoclast differentiation . Therefore, IKKβSSEE- induced osteoclastogenesis in vitro is uncoupled from inflammatory signaling with respect to TNF-α and IL-1. Nevertheless, given that IKKβSSEE does induce secretion of these factors, we must consider the possibility that this kinase modulates osteoclast activation at sites of inflammation through inflammatory signals, the nature of which will be investigated in future studies.NF-κB is also a critical regulator of inflammatory signals, and inflSSEE is sufficient for the establishment of osteolysis in vivo. The clinical significance of our findings is highlighted by our observations that IKKβSSEE-induced osteoclastogenesis is refractory to intervention with OPG and deletion of RANK/RANKL. In this regard, a number of conditions in human patients are associated with heightened bone turnover in the setting of inflammation for which a cause has not been identified.(Consistent with our in vitro findings, adenoviral gene-transfer experiments revealed that IKKβentified. Given thOur data highlight the critical role of IKKβ in osteoclast differentiation and osteolysis. We have found that constitutively active IKKβ unfolds the osteoclast program in the absence of upstream signals. We report the first evidence of RANK-independent osteoclast differentiation that is induced through a single kinase, and we propose that hyperactivation of human IKKβ may lead to diseases resulting in bone destruction that would be refractory to treatments targeting receptor–proximal signaling molecules.