ASIC3 roles in mechanosensitive elongation of nucleus pulposus cells.

Morphological changes of the nucleus pulposus (NP) cells occur concomitantly as part of the intervertebral disc (IVD) degeneration and excessive mechanical loading has been speculated as a significant key factor for contributing to such morphological changes. Therefore, we hypothesize that stress exerted on NP cells can cause a deformity of nucleus in response. The changes of cell morphology is observed in degenerative nucleus pulposus. One of the reasons for degeneration of NP is due to overloading of NP especially in the obese population. So the nucleus deformity caused by stress/force is of our study interest. To delineate the effects and role of mechanical stress, we developed a 3D assay using hydrogel cultures with a circular hole generated with needle indentation to simulate a local stress concentration along the edge of the hole. A stressed zone, encompassing 100 µm of range from the circular edge, is defined based on stress concentration calculation to enable quantitative analysis against the control zone. Our results demonstrated that the circular hole produces stress-induced morphological changes in NP cells. The tangential elongation of NP cells and their nucleus shape changes in the stressed zone are significantly increased compared to the non-stressed control zone. It is proposed that the cell elongation is a direct response to elevated stress within the stressed zone. Subsequently we found the stress induced morphological changes of the NP cells can be significantly reduced by inhibiting ASIC3. This suggests ASIC3 plays an important role of play in mechano-signaling of NP cells.

Activation of Mechanosensitive Ion Channels by Ultrasound.

Mechanosensitive channels (MSCs) play an important role in how cells transduce mechanical stimuli into electrical or chemical signals, which provides an interventional possibility through the manipulation of ion channel activation using different mechanical stimulation conditions. With good spatial resolution and depth of penetration, ultrasound is often proposed as the tool of choice for such therapeutic applications. Despite the identification of many ion channels as mechanosensitive in recent years, only a limited number of MSCs have been reported to be activated by ultrasound with substantial evidence. Furthermore, although many therapeutic implications using ultrasound have been explored, few offered insights into the molecular basis and the biological effects induced by ultrasound in relieving pain and accelerate tissue healing. In this review, we examined the literature, in particular studies that provided evidence of cellular responses to ultrasound, with and without the target ion channels. The ultrasound activation conditions were then summarized for these ion channels, and these conditions were related to their mode of activation based on the current biological concepts. The overall goal is to bridge the results relating to the activation of MSCs that is specific for ultrasound with the current knowledge in molecular structure and the available physiological evidence that may have facilitated such phenomena. We discussed how collating the information revealed by available scientific investigations helps in the design of a more effective stimulus device for the proposed translational purposes. Traditionally, studies on the effects of ultrasound have focused largely on its mechanical and physical interaction with the targeted tissue through thermal-based therapies as well as non-thermal mechanisms including ultrasonic cavitation; gas body activation; the direct action of the compressional, tensile and shear stresses; radiation force; and acoustic streaming. However, the current review explores and attempts to establish whether the application of low-intensity ultrasound may be associated with the activation of specific MSCs, which in turn triggers relevant cell signaling as its molecular mechanism in achieving the desired therapeutic effects. Non-invasive brain stimulation has recently become an area of intense research interest for rehabilitation, and the implication of low-intensity ultrasound is particularly critical given the need to minimize heat generation to preserve tissue integrity for such applications.

Auditory independent low-intensity ultrasound stimulation of mouse brain is associated with neuronal ERK phosphorylation and an increase of Tbr2 marked neuroprogenitors.

Transcranial ultrasound stimulation is an emerging technique for the development of a non-invasive neuromodulation device for the treatment of various types of neurodegenerations and brain damages. However, there are very few studies that have quantified the optimal ultrasound dosage and the long-term associated effects of transcranial ultrasound treatments of brain diseases. In this study, we used a simple ex vivo hippocampal tissues stimulated by different dosages of ultrasound in combination with different chemical treatments to quantify the required energy for a measurable effect. After determining the most desirable ex vivo stimulation conditions, it was then replicated for the in vivo mouse brains. It was discovered that transcranial ultrasound promoted the increase of Tbr2-expressing neural progenitors in an ASIC1a-dependent manner. Furthermore, such effect was observable at least a week after the initial ultrasound treatments and was not abolished by auditory toxicity.

Piezoelectric effect stimulates the rearrangement of chondrogenic cells and alters ciliary orientation via atypical PKCζ.

Therapeutic ultrasound was administered to patients suffering from bone fracture with FDA approval. Bone and cartilage are piezoelectric materials. To investigate the effects of piezoelectricity on the cells of chondrogenic lineage, we applied ultrasound stimulation on an AT-cut quartz coverslip to generate electric field fluctuations. The bone-marrow-derived mesenchymal stem cells (BMMSC) and primary chondrocytes were cultured on either glass or quartz coverslips for ultrasound stimulation. The cells were immunofluorescent-labeled for the assessment of cell arrangement and ciliary orientation. Ultrasound and piezoelectricity both stimulate cell migration and disrupt ciliary orientation induced by directional migration. In particular, piezoelectric effects on cell rearrangement can be abolished by the inhibitor specifically targeting atypical Protein kinase C zeta (PKCζ). Our findings shed light on the possibility of cellular modulation by using piezoelectric manipulation.

Dynamic Pressure Stimulation Upregulates Collagen II and Aggrecan in Nucleus Pulposus Cells Through Calcium Signaling.

STUDY DESIGN: An in vitro study to investigate the effect of pressure stimulation on nucleus pulposus (NP) cells. OBJECTIVE: The aim of this study was to investigate the question whether physical stimulation can be leveraged to enhance extracellular matrix (ECM) synthesis as a preventive measure for intervertebral disc (IVD) degeneration. SUMMARY OF BACKGROUND DATA: ECM plays an important role in regulating hydration and pressure balance of the IVD. METHODS: Cellular stimulation devices with different pressurizing protocols were used to create a pressurized environment to cells cultures. The setup was used to mimic the pressurized conditions within IVD to investigate the effect of pressure stimulation on NP cells. RESULTS: Pressure stimulation at 300 kPa can enhance the synthesis of ECM proteins Collagen II and aggrecan in NP cells and the effect of dynamic pressure stimulation outperformed the static one. The difference between static and dynamic pressure stimulation was due primarily to calcium signaling activated by pressure fluctuation. The superior effect of dynamic pressure holds for a wide range of stimulation durations, relating to the range of spontaneous calcium oscillations in NP cells. CONCLUSION: The results link mechanotransduction to the downstream ECM protein synthesis and suggest slow exercises that correspond with spontaneous calcium oscillations in NP cells can be effective to stimulate ECM synthesis in IVD.

ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain.

Accumulating evidence has shown transcranial low-intensity ultrasound can be potentially a non-invasive neural modulation tool to treat brain diseases. However, the underlying mechanism remains elusive and the majority of studies on animal models applying rather high-intensity ultrasound that cannot be safely used in humans. Here, we showed low-intensity ultrasound was able to activate neurons in the mouse brain and repeated ultrasound stimulation resulted in adult neurogenesis in specific brain regions. In vitro calcium imaging studies showed that a specific ultrasound stimulation mode, which combined with both ultrasound-induced pressure and acoustic streaming mechanotransduction, is required to activate cultured cortical neurons. ASIC1a and cytoskeletal proteins were involved in the low-intensity ultrasound-mediated mechanotransduction and cultured neuron activation, which was inhibited by ASIC1a blockade and cytoskeleton-modified agents. In contrast, the inhibition of mechanical-sensitive channels involved in bilayer-model mechanotransduction like Piezo or TRP proteins did not repress the ultrasound-mediated neuronal activation as efficiently. The ASIC1a-mediated ultrasound effects in mouse brain such as immediate response of ERK phosphorylation and DCX marked neurogenesis were statistically significantly compromised by ASIC1a gene deletion. Collated data suggest that ASIC1a is the molecular determinant involved in the mechano-signaling of low-intensity ultrasound that modulates neural activation in mouse brain.

Elevation of Intra-Cellular Calcium in Nucleus Pulposus Cells with Micro-Pipette-Guided Ultrasound.

Modulation of intra-cellular calcium by ultrasound offers a possible means for therapeutic applications. One such possibility is the modulation of nucleus pulposus cells as a preventive measure for inter-vertebral disc degeneration. We report a cellular stimulation device (micro-pipette ultrasound) using a glass micro-pipette as a waveguide to deliver ultrasound through the pipette tip and to elevate intra-cellular calcium in nucleus pulposus cells. The device generates two relevant stimuli at the cellular level: ultrasound propagation throughout the cell and acoustic streaming on the apical side. Ultrasound is radiated from a tip of a few microns, and its amplitude is proportional to the input voltage; acoustic streaming can be controlled by the duty factor. The novelty of the device is to impose a unique cellular loading: shear stress on cell apical surfaces combined with compressional waves propagating through the cells. G protein-coupled receptors and acid-sensing ion channel 3 were shown to play a role in calcium elevation by micro-pipette ultrasound in nucleus pulposus cells. Our results demonstrate that micro-pipette ultrasound can be an effective tool to elevate intra-cellular calcium levels in different cells, facilitating the identification of different mechanoreceptors in action.

Low intensity ultrasound enhances cisplatin uptake in vitro by cochlear hair cells.

Drug delivery to the inner ear has been challenging due to the blood-labyrinth barrier. Intracochlear drug delivery is an invasive alternative with less pharmacokinetic variables. In this study, the effect of low intensity ultrasound on drug uptake by hair cells is investigated. Cochlear explants harvested from newborn mice were cultured in a medium containing cisplatin to emulate drug delivered to the endolymph. The results demonstrated the exposure to ultrasound stimulation effectively enhanced cisplatin uptake by hair cells. The uptake started from the apical side of the hair cells and progressed inward as the exposure time increased.

The responses of nucleus pulposus cells to pressure and ultrasound stimulation.

A cellular stimulation device with a pressurized chamber is developed to investigate the effect of ultrasound and pressure fluctuation on nucleus pulposus (NP) cells. The pressurized chamber is designed to emulate the in vivo environment of intervertebral discs, which are under dynamic pressure, and to emulate impact during sports and exercise. Both hydrostatic pressure and ultrasound stimulation increase phosphorylation of ERK (pERK) in NP cells, and promote its translocation into nucleus. This increase in pERK levels might be activated through calcium signaling pathways as intracellular calcium in NP cells was strongly elevated by pressure changes.

Piezoelectric stimulation by ultrasound facilitates chondrogenesis of mesenchymal stem cells.

A cellular stimulation device utilizing an AT-cut quartz coverslip mounted on an ultrasonic live imaging chamber is developed to investigate the effect of piezoelectric stimulation. Two types of chambers deliver ultrasound at intensities ranging from 1 to 20 mW/cm2 to mesenchymal stem cells (MSCs) seeded on the quartz coverslip. The quartz coverslip imposes additionally localized electric charges as it vibrates with the stimulation. The device was applied to explore whether piezoelectric stimulation can facilitate chondrogenesis of MSCs. The results suggest piezoelectric stimulation drove clustering of MSCs and consequently facilitated chondrogenesis of MSCs without the use of differentiation media.

Low Intensity Ultrasound Induces Epithelial Cell Adhesion Responses.

In this study, we investigated the cellular mechanosensitive responses to a low intensity ultrasound (LIUS) stimulation (ISATA = 1 mW/cm2, pressure = 10 kPa). The dose and temporal effects at cell-substrate adhesion (CSA) at the basal level and cell-cell adhesion (CCA) at the apical level are reported in detail. A model of mouse mammary gland epithelial cells (EpH4) and the phosphorylation of mechanosensitive 130 kDa Crk-associated substrate (p130CAS) as an indicator for cellular responses were used. The intensity of phospho-p130CAS was found to be dependent on LIUS stress level, and the p130CAS was phosphorylated after 1 min stimulation at CSA. The phospho-p130CAS was also found to increase significantly at CCA upon LIUS stimulation. We confirmed that the cellular responses to ultrasound are immediate and dose dependent. Ultrasound affects not only CSA but also CCA. An E-cadherin knockout (EpH4ECad-/-) model also confirmed that phosphorylation of p130CAS at CCA is related to E-cadherins.

Primary cilia control cell alignment and patterning in bone development via ceramide-PKCζ-ß-catenin signaling.

Intraflagellar transport (IFT) proteins are essential for cilia assembly and function. IFT protein mutations lead to ciliopathies, which manifest as variable skeletal abnormalities. However, how IFT proteins regulate cell alignment during bone development is unknown. Here, we show that the deletion of IFT20 in osteoblast lineage using Osterix-Cre and inducible type I Collagen-CreERT cause a compromised cell alignment and a reduced bone mass. This finding was validated by the disorganized collagen fibrils and decreased bone strength and stiffness in IFT20-deficient femurs. IFT20 maintains cilia and cell alignment in osteoblasts, as the concentric organization of three-dimensional spheroids was disrupted by IFT20 deletion. Mechanistically, IFT20 interacts with the ceramide-PKCζ complex to promote PKCζ phosphorylation in cilia and induce the apical localization of ß-catenin in osteoblasts, both of which were disrupted in the absence of IFT20. These results reveal that IFT20 regulates polarity and cell alignment via ceramide-pPKCζ-ß-catenin signaling during bone development.

Design of an ultrasound chamber for cellular excitation and observation.

In this work, a design of integrating ultrasonic transduction with live cell imaging chamber is introduced. The principle of a metal-incident-glass-output acoustic path was used to deliver a uniform energy profile into the imaging/incubation chamber in the form of leaky Lamb waves. The design was applied to examine living mouse mammary gland epithelial cells (EpH4). Significant changes in intracellular activities were observed even at a very low energy intensity level (1 MHz, ISATA = 1 mW/cm2, continuous wave). Live imaging with ultrasonic stimulation provides a different paradigm to interrogate cellular mechanosensitive responses in real time.

Regulator of G Protein Signaling Protein 12 (Rgs12) Controls Mouse Osteoblast Differentiation via Calcium Channel/Oscillation and Gαi-ERK Signaling.

Bone homeostasis intimately relies on the balance between osteoblasts (OBs) and osteoclasts (OCs). Our previous studies have revealed that regulator of G protein signaling protein 12 (Rgs12), the largest protein in the Rgs super family, is essential for osteoclastogenesis from hematopoietic cells and OC precursors. However, how Rgs12 regulates OB differentiation and function is still unknown. To understand that, we generated an OB-targeted Rgs12 conditional knockout (CKO) mice model by crossing Rgs12fl/fl mice with Osterix (Osx)-Cre transgenic mice. We found that Rgs12 was highly expressed in both OB precursor cells (OPCs) and OBs of wild-type (WT) mice, and gradually increased during OB differentiation, whereas Rgs12-CKO mice (OsxCre/+ ; Rgs12fl/fl ) exhibited a dramatic decrease in both trabecular and cortical bone mass, with reduced numbers of OBs and increased apoptotic cell population. Loss of Rgs12 in OPCs in vitro significantly inhibited OB differentiation and the expression of OB marker genes, resulting in suppression of OB maturation and mineralization. Further mechanism study showed that deletion of Rgs12 in OPCs significantly inhibited guanosine triphosphatase (GTPase) activity and cyclic adenosine monophosphate (cAMP) level, and impaired Calcium (Ca2+ ) oscillations via restraints of major Ca2+ entry sources (extracellular Ca2+ influx and intracellular Ca2+ release from endoplasmic reticulum), partially contributed by the blockage of L-type Ca2+ channel mediated Ca2+ influx. Downstream mediator extracellular signal-related protein kinase (ERK) was found inactive in OBs of OsxCre/+ ; Rgs12fl/fl mice and in OPCs after Rgs12 deletion, whereas application of pertussis toxin (PTX) or overexpression of Rgs12 could rescue the defective OB differentiation via restoration of ERK phosphorylation. Our findings reveal that Rgs12 is an important regulator during osteogenesis and highlight Rgs12 as a potential therapeutic target for bone disorders. © 2018 American Society for Bone and Mineral Research.

Epithelial-mesenchymal transitions: insights from development.

Epithelial-mesenchymal transition (EMT) is a crucial, evolutionarily conserved process that occurs during development and is essential for shaping embryos. Also implicated in cancer, this morphological transition is executed through multiple mechanisms in different contexts, and studies suggest that the molecular programs governing EMT, albeit still enigmatic, are embedded within developmental programs that regulate specification and differentiation. As we review here, knowledge garnered from studies of EMT during gastrulation, neural crest delamination and heart formation have furthered our understanding of tumor progression and metastasis.

Alternative path to EMT: regulation of apicobasal polarity in Drosophila.

The epithelial-to-mesenchymal transition (EMT), a key process in morphogenesis, is often driven by repressing expression of adherens junction components, such as E-cadherin. In this issue of Developmental Cell, Campbell et al. (2011) uncover an alternative mechanism in the Drosophila embryonic gut that promotes EMT via Serpent, a GATA transcriptional repressor of the apicobasal polarity gene crumbs.

Target cell movement in tumor and cardiovascular diseases based on the epithelial-mesenchymal transition concept.

Epithelial-mesenchymal transition (EMT) is a fundamental mechanism in development driving body plan formation. EMT describes a transition process wherein polarized epithelial cells lose their characteristics and acquire a mesenchymal phenotype. The apico-basal polarity of epithelial cells is replaced by a front-rear polarity in mesenchymal cells which favor cell-extracellular matrix than intercellular adhesion. These events serve as a prerequisite to the context-dependent migratory and invasive functions of mesenchymal cells. In solid tumors, carcinoma cells undergoing EMT not only invade and metastasize but also exhibit cancer stem cell-like properties, providing resistance to conventional and targeted therapies. In cardiovascular systems, epicardial cells engaged in EMT contribute to myocardial regeneration. Conversely, cardiovascular endothelial cells undergoing EMT cause cardiac fibrosis. Growing evidence has shed light on the potential development of novel therapeutics that target cell movement by applying the EMT concept, and this may provide new therapeutic strategies for the treatment of cancer and heart diseases.

Essential role of Pin1 in the regulation of TRF1 stability and telomere maintenance.

Telomeres are essential for maintaining cellular proliferative capacity and their loss has been implicated in ageing. A key regulator in telomere maintenance is the telomeric protein TRF1, which was also identified as Pin2 in a screen for Pin1. Pin1 is a unique prolyl isomerase that regulates protein conformation and function after phosphorylation. However, little is known about the role of Pin1 in telomere regulation or the modulation of TRF1 by upstream signals. Here we identify TRF1 as a major conserved substrate for Pin1 during telomere maintenance and ageing. Pin1 inhibition renders TRF1 resistant to protein degradation, enhances TRF1 binding to telomeres, and leads to gradual telomere loss in human cells and in mice. Pin1-deficient mice also show widespread premature ageing phenotypes within just one generation, similar to those in telomerase-deficient mice after 4-5 consecutive generations. Thus, Pin1 is an essential regulator of TRF1 stability, telomere maintenance and ageing.

Pin1 has opposite effects on wild-type and P301L tau stability and tauopathy.

Tau pathology is a hallmark of many neurodegenerative diseases including Alzheimer disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Genetic tau mutations can cause FTDP-17, and mice overexpressing tau mutants such as P301L tau are used as AD models. However, since no tau mutations are found in AD, it remains unclear how appropriate tau mutant mice are as an AD model. The prolyl isomerase Pin1 binds and isomerizes tau and has been implicated in protecting against neurodegeneration, but whether such Pin1 regulation is affected by tau mutations is unknown. Consistent with earlier findings that Pin1 KO induces tauopathy, here we demonstrate that Pin1 knockdown or KO increased WT tau protein stability in vitro and in mice and that Pin1 overexpression suppressed the tauopathy phenotype in WT tau transgenic mice. Unexpectedly, Pin1 knockdown or KO decreased P301L tau protein stability and abolished its robust tauopathy phenotype in mice. In contrast, Pin1 overexpression exacerbated the tauopathy phenotype in P301L tau mice. Thus, Pin1 has opposite effects on the tauopathy phenotype depending on whether the tau is WT or a P301L mutant, indicating the need for disease-specific therapies for tauopathies.