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We transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1 (ALDH1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.

Microstructural adaptation of bone in response to mechanical stimuli is diminished with estrogen deprivation. Here we tested in vivo whether ovariectomy (OVX) alters the acute response of osteocytes, the principal mechanosensory cells of bone, to mechanical loading in mice. We also used super resolution microscopy (Structured Illumination microscopy or SIM) in conjunction with immunohistochemistry to assess changes in the number and organization of “osteocyte mechanosomes” - complexes of Panx1 channels, P2X7 receptors and CaV3 voltage-gated Ca(2+) channels clustered around α(v)β(3) integrin foci on osteocyte processes. Third metatarsals bones of mice expressing an osteocyte-targeted genetically encoded Ca(2+) indicator (DMP1-GCaMP3) were cyclically loaded in vivo to strains from 250 to 3000 με and osteocyte intracellular Ca(2+) signaling responses were assessed in mid-diaphyses using multiphoton microscopy. The number of Ca(2+) signaling osteocytes in control mice increase monotonically with applied strain magnitude for the physiological range of strains. The relationship between the number of Ca(2+) signaling osteocytes and loading was unchanged at 2 days post-OVX. However, it was altered markedly at 28 days post-OVX. At loads up to 1000 με, there was a dramatic reduction in number of responding (i.e. Ca(2+) signaling) osteocytes; however, at higher strains the numbers of Ca(2+) signaling osteocytes were similar to control mice. OVX significantly altered the abundance, make-up and organization of osteocyte mechanosome complexes on dendritic processes. Numbers of α(v)β(3) foci also staining with either Panx 1, P2X7R or CaV3 declined by nearly half after OVX, pointing to a loss of osteocyte mechanosomes on the dendritic processes with estrogen depletion. At the same time, the areas of the remaining foci that stained for α(v)β(3) and channel proteins increased significantly, a redistribution of mechanosome components suggesting a potential compensatory response. These results demonstrate that the deleterious effects of estrogen depletion on skeletal mechanical adaptation appear at the level of mechanosensation; osteocytes lose the ability to sense small (physiological) mechanical stimuli. This decline may result at least partly from changes in the structure and organization of osteocyte mechanosomes, which contribute to the distinctive sensitivity of osteocytes (particularly their dendritic processes) to mechanical stimulation.

Osteocytes are considered to be the major mechanosensory cells of bone, but how osteocytes in vivo process, perceive, and respond to mechanical loading remains poorly understood. Intracellular calcium (Ca2+) signaling resulting from mechanical stimulation has been widely studied in osteocytes in vitro and in bone explants, but has yet to be examined in vivo. This is achieved herein by using a three-point bending device which is capable of delivering well-defined mechanical loads to metatarsal bones of living mice while simultaneously monitoring the intracellular Ca2+ responses of individual osteocytes by using a genetically encoded fluorescent Ca2+ indicator. Osteocyte responses are imaged by using multiphoton fluorescence microscopy. We investigated the in vivo responses of osteocytes to strains ranging from 250 to 3,000 [Formula: see text] and frequencies from 0.5 to 2 Hz, which are characteristic of physiological conditions reported for bone. At all loading frequencies examined, the number of responding osteocytes increased strongly with applied strain magnitude. However, Ca2+ intensity within responding osteocytes did not change significantly with physiological loading magnitudes. Our studies offer a glimpse into how these critical bone cells respond to mechanical load in vivo, as well as provide a technique to determine how the cells encode magnitude and frequency of loading.

Background Distension of hollow organs is known to release adenosine triphosphate (ATP) from the lining epithelium, which triggers local responses and activates sensory nerves to convey information to the central nervous system. However, little is known regarding participation of ATP and mediators of ATP release, such as Pannexin 1 (Panx1) channels, in mechanisms of vaginal mechanosensory transduction and of changes imposed by diabetes and menopause, conditions associated with vaginal dysfunction and risk for impaired genital arousal. Aim To investigate if intravaginal mechanical stimulation triggers vaginal ATP release and if (a) this response involves Panx1 channels and (b) this response is altered in animal models of diabetes and menopause. Methods Diabetic Akita female mice were used as a type 1 diabetes (T1D) model and surgical castration (ovariectomy [OVX]) as a menopause model. Panx1-null mice were used to evaluate Panx1 participation in mechanosensitive vaginal ATP release. Vaginal washes were collected from anesthetized mice at baseline (non-stimulated) and at 5 minutes after intravaginal stimulation. For the OVX and Sham groups, samples were collected before surgery and at 4, 12, 22, 24, and 28 weeks after surgery. ATP levels in vaginal washes were measured using the luciferin-luciferase assay. Panx1 mRNA levels in vaginal epithelium were quantified by quantitative polymerase chain reaction. Outcomes The main outcome measures are quantification of mechanosensitive vaginal ATP release and evaluation of impact of Panx1 deletion, OVX, and T1D on this response. Results Intravaginal mechanical stimulation–induced vaginal ATP release was 84% lower in Panx1-null (P Clinical Translation Panx1 downregulation and consequent attenuation of mechanosensitive vaginal responses may be implicated in mechanisms of female genital arousal disorder, thereby providing potential targets for novel therapies to manage this condition. Strengths & Limitations Using animal models, we demonstrated Panx1 involvement in mechanosensitive vaginal ATP release and effects of T1D and menopause on this response and on Panx1 expression. A limitation is that sex steroid hormone levels were not measured, precluding correlations and insights into mechanisms that may regulate Panx1 expression in the vaginal epithelium. Conclusions Panx1 channel is a component of the vaginal epithelial mechanosensory transduction system that is essential for proper vaginal response to mechanical stimulation and is targeted in T1D and menopause.

This review focuses on bone mechanobiology in type 1 diabetes (T1D), an area of research on diabetes-associated skeletal complications that is still in its infancy. We first provide a brief overview of the deleterious effects of diabetes on the skeleton and of the knowledge gained from studies with rodent models of T1D. Second, we discuss two specific hallmarks of T1D, low insulin and high glucose, and address the extent to which they affect skeletal health. Third, we highlight the mechanosensitive nature of bone tissue and the importance of mechanical loading for bone health. We also summarize recent advances in bone mechanobiology that implicate osteocytes as the mechanosensors and major regulatory cells in the bone. Finally, we discuss recent evidence indicating that the diabetic bone is "deaf" to mechanical loading and that osteocytes are central players in mechanisms that lead to bone loss in T1D.

Osteoarthritis (OA) pathogenesis is mediated largely through the actions of proteolytic enzymes such as matrix metalloproteinase (MMP) 13. The transcriptional regulator CITED2, which suppresses the expression of MMP13 in chondrocytes, is induced by interleukin (IL)-4 in T cells and macrophages, and by moderate mechanical loading in chondrocytes. We tested the hypothesis that CITED2 mediates cross-talk between IL-4 signaling and mechanical loading-induced pathways that result in chondroprotection, at least in part, by downregulating MMP13. IL-4 induced CITED2 gene expression in human chondrocytes in a dose- and time-dependent manner through JAK/STAT signaling. Mechanical loading combined with IL-4 resulted in additive effects on inducing CITED2 expression and downregulating of MMP13 in human chondrocytes in vitro. In vivo, IL-4 gene knockout (KO) mice exhibited reduced basal levels of CITED2 expression in chondrocytes. While moderate treadmill running induced CITED2 expression and reduced MMP13 expression in wild-type mice, these effects were blunted (for CITED2) or abolished (for MMP13) in chondrocytes of IL-4 gene KO mice. Moreover, intra-articular injections of mouse recombinant IL-4 combined with regular cage activity mitigated post-traumatic OA to a greater degree compared to immobilized mice treated with IL-4 alone. These data suggest that using moderate loading to enhance IL-4 may be a potential therapeutic strategy for chondroprotection in OA.

The pannexin 1 (Panx1) channel is a mechanosensitive channel that interacts with P2X7 receptors (P2X7R) to form a functional complex that has been shown in vitro to play an essential role in osteocyte mechanosignaling. While the participation of P2X7R in skeletal responses to mechanical loading has been demonstrated, the role of Panx1 and of its interplay with P2X7R still remain to be determined. In this study, we use a global Panx1(−/−) knockout mouse model and in vivo mechanical loading to demonstrate for the first time that Panx1 channels play an essential role in load-induced skeletal responses. We found that absence of Panx1 not only disrupts the P2X7R-Panx1 signaling complex, but also alters load-induced regulation of P2X7R expression. Moreover, lack of Panx1 completely abolished load-induced periosteal bone formation. Load-induced regulation of β-catenin and sclerostin expression was dysregulated in Panx1(−/−) compared to wildtype bone. This finding suggests that Panx1 deficiency disrupts Wnt/β-catenin signaling by lowering β-catenin while favoring inhibition of bone formation by increasing load-induced sclerostin expression. In sum, this study demonstrates the existence of a Panx1-dependent mechanosensitive mechanism that not only modulates ATP signaling but also coordinates Wnt/β-catenin signaling that is essential for proper skeletal response to mechanical loading.

Osteocyte apoptosis is required to induce intracortical bone remodeling after microdamage in animal models, but how apoptotic osteocytes signal neighboring "bystander" cells to initiate the remodeling process is unknown. Apoptosis has been shown to open pannexin-1 (Panx1) channels to release adenosine diphosphate (ATP) as a "find-me" signal for phagocytic cells. To address whether apoptotic osteocytes use this signaling mechanism, we adapted the rat ulnar fatigue-loading model to reproducibly introduce microdamage into mouse cortical bone and measured subsequent changes in osteocyte apoptosis, receptor activator of NF-κB ligand (RANKL) expression and osteoclastic bone resorption in wild-type (WT; C57Bl/6) mice and in mice genetically deficient in Panx1 (Panx1KO). Mouse ulnar loading produced linear microcracks comparable in number and location to the rat model. WT mice showed increased osteocyte apoptosis and RANKL expression at microdamage sites at 3 days after loading and increased intracortical remodeling and endocortical tunneling at day 14. With fatigue, Panx1KO mice exhibited levels of microdamage and osteocyte apoptosis identical to WT mice. However, they did not upregulate RANKL in bystander osteocytes or initiate resorption. Panx1 interacts with P2X7 R in ATP release; thus, we examined P2X7 R-deficient mice and WT mice treated with P2X7 R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find-me signal. P2X7 RKO mice failed to upregulate RANKL in osteocytes or induce resorption despite normally elevated osteocyte apoptosis after fatigue loading. Similarly, treatment of fatigued C57Bl/6 mice with BBG mimicked behavior of both Panx1KO and P2X7 RKO mice; BBG had no effect on osteocyte apoptosis in fatigued bone but completely prevented increases in bystander osteocyte RANKL expression and attenuated activation of resorption by more than 50%. These results indicate that activation of Panx1 and P2X7 R are required for apoptotic osteocytes in fatigued bone to trigger RANKL production in neighboring bystander osteocytes and implicate ATP as an essential signal mediating this process.

Chaperone-mediated autophagy (CMA), a cellular process that contributes to protein quality control through targeting of a subset of cytosolic proteins to lysosomes for degradation, undergoes a functional decline with age. We have used a mouse model with liver-specific defective CMA to identify changes in proteostasis attributable to reduced CMA activity in this organ with age. We have found that other proteolytic systems compensate for CMA loss in young mice which helps to preserve proteostasis. However, these compensatory responses are not sufficient for protection against proteotoxicity induced by stress (oxidative stress, lipid challenges) or associated with aging. Livers from old mice with CMA blockage exhibit altered protein homeostasis, enhanced susceptibility to oxidative stress and hepatic dysfunction manifested by a diminished ability to metabolize drugs, and a worsening of the metabolic dysregulation identified in young mice. Our study reveals that while the regulatory function of CMA cannot be compensated for in young organisms, its contribution to protein homeostasis can be handled by other proteolytic systems. However, the decline in the compensatory ability identified with age explains the more severe consequences of CMA impairment in older organisms and the contribution of CMA malfunction to the gradual decline in proteostasis and stress resistance observed during aging.

Osteocyte processes are an order of magnitude more sensitive to mechanical loading than their cell bodies. The mechanisms underlying this remarkable mechanosensitivity are not clear, but may be related to the infrequent αVβ3 integrin sites where the osteocyte cell processes attach to canalicular walls. These sites develop dramatically elevated strains during load-induced fluid flow in the lacunar-canalicular system and were recently shown to be primary sites for osteocyte-like MLO-Y4 cell mechanotransduction. These αVβ3 integrin sites lack typical integrin transduction mechanisms. Rather, stimulation at these sites alters Ca2+ signaling, ATP release and membrane potential. In the current studies, we tested the hypothesis that in authentic osteocytes in situ, key membrane proteins implicated in osteocyte mechanotransduction are preferentially localized at or near to β3 integrin-foci. We analyzed these spatial relationships in mouse bone osteocytes using immunohistochemistry combined with Structured Illumination Super Resolution Microscopy, a method that permits structural resolution at near electron microscopy levels in tissue sections. We discovered that the purinergic channel pannexin1, the ATP-gated purinergic receptor P2X7R and the low voltage transiently opened T-type calcium channel CaV3.2−1 all reside in close proximity to β3 integrin attachment foci on osteocyte processes, suggesting a specialized mechanotransduction complex at these sites. We further confirmed this observation on isolated osteocytes in culture using STochasitc Optical Resonance Microscopy. These findings identify a possible structural basis for the unique mechanosensation and transduction capabilities of the osteocyte process. This article is protected by copyright. All rights reserved

Urothelial cells respond to bladder distension with ATP release, and ATP signaling within the bladder and from the bladder to the CNS is essential for proper bladder function. In other cell types, pannexin 1 (Panx1) channels provide a pathway for mechanically-induced ATP efflux and for ATP-induced ATP release through interaction with P2X7 receptors (P2X7Rs). We report that Panx1 and P2X7R are functionally expressed in the bladder mucosa and in immortalized human urothelial cells (TRT-HU1), and participate in urothelial ATP release and signaling. ATP release from isolated rat bladders induced by distention was reduced by the Panx1 channel blocker mefloquine (MFQ) and was blunted in mice lacking Panx1 or P2X7R expression. Hypoosmotic shock induced YoPro dye uptake was inhibited by MFQ and the P2X7R blocker A438079 in TRT-HU1 cells, and was also blunted in primary urothelial cells derived from mice lacking Panx1 or P2X7R expression. Rinsing-induced mechanical stimulation of TRT-HU1 cells triggered ATP release, which was reduced by MFQ and potentiated in low divalent cation solution (LDPBS), a condition known to enhance P2X7R activation. ATP signaling evaluated as intercellular Ca2+ wave radius was significantly larger in LDPBS, reduced by MFQ and by apyrase (ATP scavenger). These findings indicate that Panx1 participates in urothelial mechanotransduction and signaling by providing a direct pathway for mechanically-induced ATP release and by functionally interacting with P2X7Rs.

SummaryAging-related oxidative stress has been linked to degenerative modifications in different organs and tissues. Using redox proteomic analysis and illustrative tandem mass spectrometry mapping, we demonstrate oxidative posttranslational modifications in structural proteins of intervertebral discs (IVDs) isolated from aging mice. Increased protein carbonylation was associated with protein fragmentation and aggregation. Complementing these findings, a significant loss of elasticity and increased stiffness was measured in fibrocartilage from aging mice. Studies using circular dichroism and intrinsic tryptophan fluorescence revealed a significant loss of secondary and tertiary structures of purified collagens following oxidation. Collagen unfolding and oxidation promoted both nonenzymatic and enzymatic degradation. Importantly, induction of oxidative modification in healthy fibrocartilage recapitulated the biochemical and biophysical modifications observed in the aging IVD. Together, these results suggest that protein carbonylation, glycation, and lipoxidation could be early events in promoting IVD degenerative changes.