Insulin-like growth factor-1 regulates the mechanosensitivity of chondrocytes by modulating TRPV4.

Both mechanical and biochemical stimulation are required for maintaining the integrity of articular cartilage. However, chondrocytes respond differently to mechanical stimuli in osteoarthritic cartilage when biochemical signaling pathways, such as Insulin-like Growth Factor-1 (IGF-1), are altered. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel is central to chondrocyte mechanotransduction and regulation of cartilage homeostasis. Here, we propose that changes in IGF-1 can modulate TRPV4 channel activity. We demonstrate that physiologic levels of IGF-1 suppress hypotonic-induced TRPV4 currents and intracellular calcium flux by increasing apparent cell stiffness that correlates with actin stress fiber formation. Disruption of F-actin following IGF-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Using point mutations of the TRPV4 channel at the microtubule-associated protein 7 (MAP-7) site shows that regulation of TRPV4 by actin is mediated via the interaction of actin with the MAP-7 domain of TRPV4. We further highlight that ATP release, a down-stream response to mechanical stimulation in chondrocytes, is mediated by TRPV4 during hypotonic challenge. This response is significantly abrogated with IGF-1 treatment. As chondrocyte mechanosensitivity is greatly altered during osteoarthritis progression, IGF-1 presents as a promising candidate for prevention and treatment of articular cartilage damage.

Evaluation of multi-color genetically encoded Ca2+ indicators in filamentous fungi.

Genetically encoded Ca2+ indicators (GECIs) enable long-term monitoring of cellular and subcellular dynamics of this second messenger in response to environmental and developmental cues without relying on exogenous dyes. Continued development and optimization in GECIs, combined with advances in gene manipulation, offer new opportunities for investigating the mechanism of Ca2+ signaling in fungi, ranging from documenting Ca2+ signatures under diverse conditions and genetic backgrounds to evaluating how changes in Ca2+ signature impact calcium-binding proteins and subsequent cellular changes. Here, we attempted to express multi-color (green, yellow, blue, cyan, and red) circularly permuted fluorescent protein (FP)-based Ca2+ indicators driven by multiple fungal promoters in Fusarium oxysporum, F. graminearum, and Neurospora crassa. Several variants were successfully expressed, with GCaMP5G driven by the Magnaporthe oryzae ribosomal protein 27 and F. verticillioides elongation factor-1α gene promoters being optimal for F. graminearum and F. oxysporum, respectively. Transformants expressing GCaMP5G were compared with those expressing YC3.60, a ratiometric Cameleon Ca2+ indicator. Wild-type and three Ca2+ signaling mutants of F. graminearum expressing GCaMP5G exhibited improved signal-to-noise and increased temporal and spatial resolution and are also more amenable to studies involving multiple FPs compared to strains expressing YC3.60.

Regulation of Stem Cell Function in an Engineered Vocal Fold-Mimetic Environment.

Human mesenchymal stem cells (hMSCs) have been proposed as therapeutic cells for the treatment of vocal fold (VF) scarring. Although functional recovery was observed in animal models after stem cell injection, it is not clear how injected stem cells interact locally with the extracellular matrix (ECM) of the lamina propria (LP) and how such interactions affect stem cell behaviors to improve function. Herein, we developed an in vitro cell culture platform where hMSCs were encapsulated in a LP-mimetic matrix, derived from hyaluronic acid (HA), poly(ethylene glycol) (PEG) and collagen, and cultured dynamically in a custom-designed VF bioreactor. The cell culture system was characterized by oscillatory shear rheology, laser doppler vibrometry (LDV), and digital image correlation (DIC). A constitutive finite element analysis (FEA) model was further developed to predict vibratory responses of the hydrogel. LDV analysis demonstrated an average displacement of 47 µm in the center of the hydrogel construct at 200 Hz applied frequency without any harmonics. The predicted strains throughout the hydrogel ranged from 0 to 0.03, in good agreement with reported values for the VF. The 3D cellular construct was subjected to vibrational stimulations at 200 Hz for an optimized duration of 1 h, as confirmed by a maximal c-Fos upregulation at the transcript level. Vibrational culture over a 3-day period with a 1h-on/1h-off pattern did not compromise the overall cell viability, but resulted in a significant downregulation of fibrogenic markers and diminished staining for alpha smooth muscle actin (αSMA). Collectively, high frequency mechanical loading resulted in the loss of myofibrogenic potential and a shift away from a fibrotic phenotype.

CK2.3, a Mimetic Peptide of the BMP Type I Receptor, Increases Activity in Osteoblasts over BMP2.

Bone is one of the most important organs in the human body. It provides structure, function, and protection for other vital organs; therefore, bone maintenance and homeostasis are critical processes. As humans age, their bone mineral density decreases, which leads to diseases like osteoporosis. This disease affects one in two women and one in five men aged 50 and over. As the aging population increases, the interest and significance of studying this debilitating bone disease becomes more relevant. Current therapeutic products for osteoporosis have many side effects and can be taken for a limited number of years. Most therapeutic products only focus on decreasing bone resorption, not increasing bone formation. Bone morphogenetic protein 2 is an essential growth factor that drives osteoblast differentiation and activity and is essential for bone formation. However, usage in the clinic is unsuccessful due to several side effects. Recently, a signaling disparity in bone marrow stromal cells within the bone morphogenetic protein pathway that led to decreased bone morphogenetic protein 2 responsiveness was identified in patients diagnosed with osteoporosis. However, it is unclear how other cell populations, especially osteoblasts, which are key players in bone remodeling, are affected and whether the bone morphogenetic protein pathway is affected during osteoporosis. Our research group designed a novel peptide, casein kinase 2.3, that acts downstream of the bone morphogenetic receptor type Ia and increases bone mineralization in murine cells and primary bovine osteoblasts. The aim of the study presented here was to compare the responsiveness of osteoblasts to bone morphogenetic protein 2 and casein kinase 2.3, especially in patients diagnosed with osteoporosis. Mature osteoblasts were extracted from patients diagnosed with osteoporosis or osteoarthritis from Christiana Care Hospital in Newark, Delaware. They were stimulated with either bone morphogenetic protein 2 or casein kinase 2.3, and their effect on osteoblast activity was determined. The osteoporotic patients showed no mineralization response to bone morphogenetic protein 2 stimulation, while the osteoarthritis patients significantly responded to bone morphogenetic protein 2 stimulation. Furthermore, markers for osteoblast activity were increased by casein kinase 2.3, which was in sharp contrast to bone morphogenetic protein 2. This further supports a major bone morphogenetic protein signaling disparity in both the elderly and those suffering with osteoporosis. Both patient types did significantly respond to casein kinase 2.3. Further analysis of the bone morphogenetic protein pathway could lead to new therapeutic products for osteoporosis.

Spontaneous calcium signaling of cartilage cells: from spatiotemporal features to biophysical modeling.

Intracellular calcium ([Ca2+]i) oscillation is a fundamental signaling response of cartilage cells under mechanical loading or osmotic stress. Chondrocytes are usually considered as nonexcitable cells with no spontaneous [Ca2+]i signaling. This study proved that chondrocytes can exhibit robust spontaneous [Ca2+]i signaling without explicit external stimuli. The intensity of [Ca2+]i peaks from individual chondrocytes maintain a consistent spatiotemporal pattern, acting as a unique "fingerprint" for each cell. Statistical analysis revealed lognormal distributions of the temporal parameters of [Ca2+]i peaks, as well as strong linear correlations between their means and sds. Based on these statistical findings, we hypothesized that the spontaneous [Ca2+]i peaks may result from an autocatalytic process and that [Ca2+]i oscillation is controlled by a threshold-regulating mechanism. To test these 2 mechanisms, we established a multistage biophysical model by assuming the spontaneous [Ca2+]i signaling of chondrocytes as a combination of deterministic and stochastic processes. The theoretical model successfully explained the lognormal distribution of the temporal parameters and the fingerprint feature of [Ca2+]i peaks. In addition, by using antagonists for 10 pathways, we revealed that the initiation of spontaneous [Ca2+]i peaks in chondrocytes requires the presence of extracellular Ca2+, and that the PLC-inositol 1,4,5-trisphosphate pathway, which controls the release of calcium from the endoplasmic reticulum, can affect the initiation of spontaneous [Ca2+]i peaks in chondrocytes. The purinoceptors and transient receptor potential vanilloid 4 channels on the plasma membrane also play key roles in the spontaneous [Ca2+]i signaling of chondrocytes. In contrast, blocking the T-type or L-type voltage-gated calcium channel promoted the spontaneous calcium signaling. This study represents a systematic effort to understand the features and initiation mechanisms of spontaneous [Ca2+]i signaling in chondrocytes, which are critical for chondrocyte mechanobiology.-Zhou, Y., Lv, M., Li, T., Zhang, T., Duncan, R., Wang, L., Lu, X. L. Spontaneous calcium signaling of cartilage cells: from spatiotemporal features to biophysical modeling.

Culture of Mesenchymal Stem Cells in a Hydrogel Model of Vocal Fold Lamina Propria.

Stem cell injection has been proposed as an alternative approach for the restoration of vocal fold (VF) function in patients with VF scarring. To assess the therapeutic efficacy of this treatment strategy, we evaluated the behaviors of human mesenchymal stem cells (hMSCs) in hydrogels derived from thiolated hyaluronic acid (HA-SH) and poly(ethylene glycol) diacrylate (PEG-DA) entrapping assembled collagen fibrils (abbreviated as HPC gels). Three hydrogel formulations with varying amounts of collagen (0, 1 and 2 mg/mL) but a fixed HA-SH (5 mg/mL) and PEG-DA (2 mg/mL) concentration, designated as HPC0, HPC1 and HPC2, were investigated. The HPC gels exhibit similar pore sizes (35-50 nm) and AFM indentation moduli (~175 Pa), although the elastic shear modulus for HPC1 (~32 Pa) is lower than HPC0 and HPC2 (~55 Pa). Although HPC1 and HPC2 gels both promoted the development of an elongated cell morphology, greater cell spreading was observed in HPC2 than in HPC1 by day 7. At the transcript level, cells cultured in HPC1 and HPC2 gels had an increased expression of fibronectin and integrin ß1, but a decreased expression of tissue inhibitor of metalloproteinase-1, collagen types I/III and HA synthase-1 when compared to cells cultured in HPC0 gels. Cellular expression of connective tissue growth factor was also elevated in HPC1 and HPC2 cultures. Importantly, the HPC2 hydrogels promoted a signficant up-regulation of matrix metalloproteinase 1, transforming growth factor ß1, and epithelial growth factor receptor, indicating an increased tissue turnover. Overall, hMSCs cultured in HPC2 gels adopt a phenotype reminiscent of cells involved in the wound healing process, providing a platform to study the effectiveness of therapeutic stem cell treatments for VF scarring.

Synthetic Peptide CK2.3 Enhances Bone Mineral Density in Senile Mice.

BACKGROUND: Osteoporosis is a silent disease caused by low bone mineral density that results in bone fractures in 1 out of 2 women and 1 in 4 men over the age of 50. Although several treatments for osteopenia and osteoporosis are available, they have severe side effects and new treatments are desperately needed. Current treatments usually target osteoclasts and inhibit their activity or differentiation. Treatments that decrease osteoclast differentiation and activity but enhance osteogenesis and osteoblast activity are not available. We recently developed a peptide, CK2.3, that induces bone formation and increases bone mineral density as demonstrated by injection over the calvaria of 6 to 9-day-old mice and tail vein injection of 8-week-old mice. CK2.3 also decreased osteoclast formation and activity. However, these studies raise questions: does CK2.3 induce similar results in old mice and if so, what is the effective CK2.3 concentration and, is the bone mineral density of vertebrae of the spinal column increased as well? METHODS: CK2.3 was systematically injected into the tail vein of female 6-month old mice with various concentrations of CK2.3: 0.76 µg/kg, 2.3 µg/kg, or 6.9 µg/kg per mice. Mice were sacrificed one week, two weeks, and four weeks after the first injection. Their spines and femurs were collected and analyzed for bone formation. RESULTS: Femur and lumbar spine analyses found increased bone mineral density (BMD) and mineral apposition rate, with greater stiffness observed in femoral samples four weeks after the first injection. Histochemistry showed that osteoclastogenesis was suppressed in CK2.3 treated senile mice. CONCLUSIONS: For the first time, this study showed the increase of lumbar spine BMD by CK2.3. Moreover, it showed that enhancement of femur BMD was accompanied by increased femur stiffness only at medium concentration of CK2.3 four weeks after the first injection indicating the maintenance of bone's structural integrity by CK2.3.

Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3.

Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone densityover time. It affects one in two women and one in four men, age 50 and older. New treatmentsthat specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, thatacts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesisin-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cellsremains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3to Quantum Dot®s (Qdot®s), semiconductor nanoparticles. We purified CK2.3-Qdot®s by sizeexclusion chromatography and verified the conjugation and stability using UV/VIS and Fouriertransform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot®s andthe conjugate was stable for at least 4 days at 37 °C. Moreover, CK2.3-Qdot®s exerted biologicalresponse similar to CK2.3. Addition of CK2.3-Qdot®s to cells followed by confocal imaging revealedthat CK2.3-Qdot®s were internalized at 6 h post stimulation. Furthermore, using pharmacologicalinhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot®s were internalized bycaveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cellthrough caveolae mediated endocytosis.

The Preservation of Bone Cell Viability in a Human Femoral Head through a Perfusion Bioreactor.

Current methods for drug development and discovery involve pre-clinical analyses that are extremely expensive and time consuming. Animal models are not the best precedent to use, when comparing to human models as they are not synonymous with the human response, thus, alternative methods for drug development are needed. One of which could be the use of an ex vivo human organ where drugs could be tested and the effects of those drugs could be observed. Finding a viable human organ to use in these preliminary ex vivo studies is difficult due to the availability, cost, and viability. Bone tissue and marrow contain a plethora of both bone and stem cells, however, these cells need constant perfusion to be viable over a longer time range. Here we maintain bone cell sustainability in an ex vivo model, through the use of human femoral heads in a novel bioreactor. This bioreactor was designed to directly perfuse cell culture media (DMEM) through the vasculature of a femoral head, providing ideal nutrients and conditions required for maintaining organ viability. We show, for the first time, that cells within a femoral head can stay alive up to 12 h. Further development could be used to determine the effects of drugs on a human organ system and could aid in the understanding of the progression of bone diseases and pathologies.

Roles of three Fusarium graminearum membrane Ca2+ channels in the formation of Ca2+ signatures, growth, development, pathogenicity and mycotoxin production.

Similar to animals and plants, external stimuli cause dynamic spatial and temporal changes of cytoplasmic Ca2+ in fungi. Such changes are referred as the Ca2+ signature and control cellular responses by modulating the activity or location of diverse Ca2+-binding proteins (CBPs) and also indirectly affecting proteins that interact with CBPs. To understand the mechanism underpinning Ca2+ signaling, therefore, characterization of how Ca2+ moves to and from the cytoplasm to create Ca2+ signatures under different conditions is fundamental. Three genes encoding plasma membrane Ca2+ channels in a Fusarium graminearum strain that expresses a fluorescent protein-based Ca2+ indicator in the cytoplasm were mutagenized to investigate their roles in the generation of Ca2+ signatures under different growth conditions and genetic backgrounds. The genes disrupted include CCH1 and MID1, which encode a high affinity Ca2+ uptake system, and FIG1, encoding a low affinity Ca2+ channel. Resulting mutants were also analyzed for growth, development, pathogenicity and mycotoxin production to determine how loss of each of the genes alters these traits. To investigate whether individual genes influence the function and expression of other genes, phenotypes and Ca2+ signatures of their double and triple mutants, as well as their expression patterns, were analyzed.

Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs.

Human cartilage endplate permeability varies with degeneration and intervertebral disc site.

Despite the critical functions the human cartilage endplate (CEP) plays in the intervertebral disc, little is known about its structural and mechanical properties and their changes with degeneration. Quantifying these changes with degeneration is important for understanding how the CEP contributes to the function and pathology of the disc. Therefore the objectives of this study were to quantify the effect of disc degeneration on human CEP mechanical properties, determine the influence of superior and inferior disc site on mechanics and composition, and simulate the role of collagen fibers in CEP and disc mechanics using a validated finite element model. Confined compression data and biochemical composition data were used in a biphasic-swelling model to calculate compressive extrafibrillar elastic and permeability properties. Tensile properties were obtained by applying published tensile test data to an ellipsoidal fiber distribution. Results showed that with degeneration CEP permeability decreased 50-60% suggesting that transport is inhibited in the degenerate disc. CEP fibers are organized parallel to the vertebrae and nucleus pulposus and may contribute to large shear strains (0.1-0.2) and delamination failure of the CEP commonly seen in herniated disc tissue. Fiber-reinforcement also reduces CEP axial strains thereby enhancing fluid flux by a factor of 1.8. Collectively, these results suggest that the structure and mechanics of the CEP may play critical roles in the solute transport and disc mechanics.

Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage.

Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.

Effects of Osmolarity on the Spontaneous Calcium Signaling of In Situ Juvenile and Adult Articular Chondrocytes.

Calcium is a universal second messenger that mediates the metabolic activity of chondrocytes in articular cartilage. Spontaneous intracellular calcium ([Ca(2+)]i) oscillations, similar to those in neurons and myocytes, have recently been observed in chondrocytes. This study analyzed and compared the effects of different osmotic environments (hypertonic, hypotonic, and isotonic) on the spontaneous [Ca(2+)]i signaling of in situ chondrocytes residing in juvenile and adult cartilage explants. In spite of a lower cell density, a significantly higher percentage of chondrocytes in adult cartilage under all osmotic environments demonstrated spontaneous [Ca(2+)]i oscillations than chondrocytes in juvenile cartilage. For both juvenile and adult chondrocytes, hypotonic stress increased while hypertonic stress decreased the response rates. Furthermore, the spatiotemporal characteristics of the [Ca(2+)]i peaks vary in an age-dependent manner. In the hypotonic environment, the [Ca(2+)]i oscillation frequency of responsive adult cells is almost tripled whereas the juvenile cells respond with an increased duration and magnitude of each [Ca(2+)]i peak. Both juvenile and adult chondrocytes demonstrated significantly slower [Ca(2+)]i oscillations with longer rising and recovery time under the hypertonic condition. Taken together, these results shed new insights into the interplay between age and osmotic environment that may regulate the fundamental metabolism of chondrocytes.

Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells.

Mechanical cues direct the lineage commitment of mesenchymal stem cells (MSCs). In this study, we identified the operative molecular mechanisms through which dynamic tensile loading (DL) regulates changes in chromatin organization and nuclear mechanics in MSCs. Our data show that, in the absence of exogenous differentiation factors, short term DL elicits a rapid increase in chromatin condensation, mediated by acto-myosin based cellular contractility and the activity of the histone-lysine N-methyltransferase EZH2. The resulting change in chromatin condensation stiffened the MSC nucleus, making it less deformable when stretch was applied to the cell. We also identified stretch induced ATP release and purinergic calcium signaling as a central mediator of this chromatin condensation process. Further, we showed that DL, through differential stabilization of the condensed chromatin state, established a 'mechanical memory' in these cells. That is, increasing strain levels and number of loading events led to a greater degree of chromatin condensation that persisted for longer periods of time after the cessation of loading. These data indicate that, with mechanical perturbation, MSCs develop a mechanical memory encoded in structural changes in the nucleus which may sensitize them to future mechanical loading events and define the trajectory and persistence of their lineage specification.

A mutation in TRPV4 results in altered chondrocyte calcium signaling in severe metatropic dysplasia.

Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is a polymodal modulated non-selective cation channel required for normal development and maintenance of bone and cartilage. Heterozygous mutations of this channel cause a variety of channelopathies, including metatropic dysplasia (MD). We analyzed the effect of a novel TRPV4 mutation c.2398G>A, p.Gly800Asp on intracellular calcium ([Ca(2+) ]i ) regulation in chondrocytes and compared this response to chondrocytes with a frequently observed mutation, c.2396C>T, p.Pro799Leu. We observed temperature-dependent [Ca(2+) ]i oscillations in both intact and MD chondrocytes however, MD mutations exhibited increased peak magnitudes of [Ca(2+) ]i during oscillations. We also found increased baseline [Ca(2+) ]i in MD primary cells, as well as increased [Ca(2+) ]i response to either hypotonic swelling or the TRVP4-specific agonist, GSK1016790A. Oscillations and stimulation responses were blocked with the TRPV4-specific antagonist, GSK205. Analysis of [Ca(2+) ]i response kinetics showed that MD chondrocytes had increased frequency of temperature-sensitive oscillations, and the magnitude and duration of [Ca(2+) ]i responses to given stimuli. Duration of the response of the p.Gly800Asp mutation to stimulation was greater than for the p.Pro799Leu mutation. These experiments show that this region of the channel is essential for proper [Ca(2+) ]i regulation. These studies of primary cells from patients show how both mutant and WT TRPV4 channels regulate cartilage and bone development. © 2015 Wiley Periodicals, Inc.

Roles of three Fusarium oxysporum calcium ion (Ca(2+)) channels in generating Ca(2+) signatures and controlling growth.

Spatial and temporal changes of cytoplasmic calcium ions ([Ca(2+)]c), caused by external stimuli, are known as the Ca(2+) signature and presumably control cellular and developmental responses. Multiple types of ion channels, pumps, and transporters on plasma and organellar membranes modulate influx and efflux of Ca(2+) to and from the extracellular environment and internal Ca(2+) stores to form Ca(2+) signatures. Expression of a fluorescent protein-based Ca(2+) probe, Cameleon YC3.60, in Fusarium oxysporum enabled us to study how disruption of three Ca(2+) channel genes, including FoCCH1, FoMID1 and FoYVC1, affects Ca(2+) signature formation at polarized hyphal tips and whether specific changes in the Ca(2+) signature caused by these mutations are related to growth-related phenotypes. Resulting mutants displayed altered amplitude, interval, and duration of Ca(2+) pulses under various external Ca(2+) concentrations as well as changes in sporulation and growth. Loss of FoMID1 and FoCCH1, genes encoding putative plasma membrane channel proteins, had a major impact on Ca(2+) signatures and growth, while disruption of FoYVC1, which encodes a vacuolar channel, only subtly affected both traits. Results from our study provide new insights into the underpinning of Ca(2+) signaling in fungi and its role in controlling growth and also raise several new questions.

Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes.

Voltage-sensitive calcium channels (VSCC) regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1) and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM) obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.

Experimental integrative muscular movement technique enhances cervical range of motion in patients with chronic neck pain: a pilot study.

INTRODUCTION: Neck pain presents a tremendous physical and financial burden. This study compared the efficacy of the complementary and alternative medical treatments of integrative muscular movement technique (IMMT) and Swedish massage on neck pain in women of occupation age, the largest demographic group with neck pain. METHODS: A total of 38 women were assigned to IMMT (n=28) or Swedish massage (n=10) in a blinded manner. Both groups received eight 30-minute treatments over 4 weeks. Cervical range of motion (ROM) in flexion, extension, sidebending, and rotation was measured before and after treatment. Each patient's pain was assessed by using an analogue pain scale of 0-10. RESULTS: Compared with the Swedish massage group, patients receiving IMMT experienced a significant increase in ROM in cervical flexion (p<0.001), extension (p<0.001), sidebending (p<0.05), and rotation (p<0.001). Absolute change in pain for IMMT was -1.75 units compared with -0.3 units for Swedish massage (p<0.05). CONCLUSION: Patients receiving the IMMT demonstrated significantly improved cervical ROM in every movement measured compared with Swedish massage. Inclusion of the IMMT in a treatment regimen for chronic neck pain may lead to decreased pain and increased cervical ROM. These positive effects of the IMMT intervention may have a role in enhancing functional outcomes in patients with neck pain.

Sodium-calcium exchanger 1 regulates epithelial cell migration via calcium-dependent extracellular signal-regulated kinase signaling.

Na(+)/Ca(2+) exchanger-1 (NCX1) is a major calcium extrusion mechanism in renal epithelial cells enabling the efflux of one Ca(2+) ion and the influx of three Na(+) ions. The gradient for this exchange activity is provided by Na,K-ATPase, a hetero-oligomer consisting of a catalytic α-subunit and a regulatory ß-subunit (Na,K-ß) that also functions as a motility and tumor suppressor. We showed earlier that mice with heart-specific ablation (KO) of Na,K-ß had a specific reduction in NCX1 protein and were ouabain-insensitive. Here, we demonstrate that Na,K-ß associates with NCX1 and regulates its localization to the cell surface. Madin-Darby canine kidney cells with Na,K-ß knockdown have reduced NCX1 protein and function accompanied by 2.1-fold increase in free intracellular calcium and a corresponding increase in the rate of cell migration. Increased intracellular calcium up-regulated ERK1/2 via calmodulin-dependent activation of PI3K. Both myosin light chain kinase and Rho-associated kinase acted as mediators of ERK1/2-dependent migration. Restoring NCX1 expression in ß-KD cells reduced migration rate and ERK1/2 activation, suggesting that NCX1 functions downstream of Na,K-ß in regulating cell migration. In parallel, inhibition of NCX1 by KB-R7943 in Madin-Darby canine kidney cells, LLC-PK1, and human primary renal epithelial cells (HREpiC) increased ERK1/2 activation and cell migration. This increased migration was associated with high myosin light chain phosphorylation by PI3K/ERK-dependent mechanism in HREpiC cells. These data confirm the role of NCX1 activity in regulating renal epithelial cell migration.