Search

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

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 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.

The "Bystander" and "Good Samaritan" effects involve the transfer of toxic or beneficial compounds from one cell to a generally adjacent other through gap junction channels and through extracellular routes. The variety of injuries in which bystander cell killing or protection occurs has greatly expanded in the last decade to include infectious agents and therapeutic compounds, radiation injury, chaperones in cell therapy and apoptosis in development. This has been accompanied by the appreciation that both gap junction mediated and paracrine routes are used for the signaling of the "kiss of life" and the "kiss of death" and that manipulations of these pathways and the molecules that use them may find therapeutic utility in treatment of a variety of pathological conditions.

Osteoblasts sense and respond to mechanical stimuli in a process involving influx and release of large ions and signaling molecules. Unapposed gap junction hemichannels formed of connexin43 (Cx43) have been proposed as a major route for such exchange, and in particular for release of ATP and prostaglandin E2 (PGE2) in osteocytes. However, we have found that Cx43-null osteoblasts have unaltered mechanically-induced PGE2 release and ATP-induced YoPro dye uptake. In contrast, PGE2 release in response to fluid shear stress is abolished in P2X7 receptor (P2X7R) null osteoblasts and ATP-induced dye uptake is attenuated following treatment of wildtype cells with a P2X7R or Pannexin1 (Panx1) channel blocker. These data indicate that Panx1 channels, in concert with P2X7R, likely form a molecular complex that performs the hemichannel function in osteoblast mechanosignaling.

In this review we will examine from a biomechanical and ultrastructural viewpoint how the cytoskeletal specialization of three basic cell types, endothelial cells (ECs), epithelial cells (renal tubule) and dendritic cells (osteocytes), enables the mechano-sensing of fluid flow in both their native in vivo environment and in culture, and the downstream signaling that is initiated at the molecular level in response to fluid flow. These cellular responses will be discussed in terms of basic mysteries and paradoxes encountered by each cell type. In ECs fluid shear stress (FSS) is nearly entirely attenuated by the endothelial glycocalyx that covers their apical membrane and yet FSS is communicated to both intracellular and junctional molecular components in activating a wide variety of signaling pathways. The same is true in proximal tubule (PT) cells where a dense brush border of microvilli covers the apical surface and the flow at the apical membrane is negligible. A four decade old unexplained mystery is the ability of PT epithelia to reliably reabsorb 60% of the flow entering the tubule regardless of the glomerular filtration rate. In the cortical collecting duct (CCD) the flow rates are so low that a special sensing apparatus, a primary cilia is needed to detect very small variations in tubular flow. In bone it has been a century old mystery as to how osteocytes embedded in a stiff mineralized tissue are able to sense miniscule whole tissue strains that are far smaller than the cellular level strains required to activate osteocytes in vitro.

The gap junction protein connexin43 (Cx43) has been proposed to play key roles in bone differentiation and mineralization, but underlying cellular mechanisms are not totally understood. To further explore roles of Cx43 in these processes, we immortalized calvarial osteoblasts from wild-type and Cx43-null mice using human telomerase reverse transcriptase (hTERT). Osteoblastic (MOB) cell lines were generated from three individual wild-type and three individual Cx43-null mouse calvaria. Average population doubling times of the cell lines were higher than of the primary osteoblasts but did not greatly differ with regard to genotype. Modest to high level of Cx45 expression was detected in MOBs of both genotypes. Most of the cell lines expressed osteoblastic markers [Type I collagen, osteopontin, osteocalcin, parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP), periostin (OSF-2), osterix (Osx), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP)], and mineralization was comparable to that of primary osteoblasts. Two MOB cell lines from each genotype with most robust maintenance of osteoblast lineage markers were analyzed in greater detail, revealing that the Cx43-null cell lines showed a significant delay in early differentiation (up to 9 days in culture). Matrix mineralization was markedly delayed in one of the Cx43-null lines and slightly delayed in the other. These findings comparing new and very stable wild-type and Cx43-null osteoblastic cell lines define a role for Cx43 in early differentiation and mineralization stages of osteoblasts and further support the concept that Cx43 plays important role in the cellular processes associated with skeleton function.

Fluid-induced shear stress is widely recognized as an important biophysical signal in cell-cell mechanotransduction. To identify cellular signaling pathways that are regulated by fluid shear stress, we applied the unbiased approach of transcriptional profiling. Our cDNA array analysis detected that 1,165 of the 6,288 sampled unigenes were significantly affected by pulsatile fluid flow. GenMapp 2.1 analysis revealed pathways of genes regulated by shear stress: angiogenesis, blood vessel morphogenesis, regulation of endothelial cell proliferation, and prostaglandin biosynthesis. Individual genes significantly up-/downregulated by shear stress included vascular endothelial growth factor A (Vegf a), cysteine-rich protein 61 (Cyr61), platelet-derived growth factor-alpha (Pdgf a), connective tissue growth factor (Ctgf), Neuropilin 1 (Nrp1), angiotensin II receptor, type 1 a (Agtr1 a) and fibroblast growth factor 1 (Fgf1). Based on these findings, we hypothesize that fluid shear stress-regulated Vegf most likely stimulates MC3T3-E1 cells through autocrine/paracrine release and may provide a powerful recruitment signal for osteoclasts, endothelial cells, and/or stem cells during bone remodeling.

We propose a conceptual model for the cytoskeletal organization of endothelial cells (ECs) based on a major dichotomy in structure and function at basal and apical aspects of the cells. Intracellular distributions of filamentous actin (F-actin), vinculin, paxillin, ZO-1, and Cx43 were analyzed from confocal micrographs of rat fat-pad ECs after 5 h of shear stress. With intact glycocalyx, there was severe disruption of the dense peripheral actin bands (DPABs) and migration of vinculin to cell borders under a uniform shear stress (10.5 dyne/cm 2 ; 1 dyne = 10 μN). This behavior was augmented in corner flow regions of the flow chamber where high shear stress gradients were present. In striking contrast, no such reorganization was observed if the glycocalyx was compromised. These results are explained in terms of a “bumper-car” model, in which the actin cortical web and DPAB are only loosely connected to basal attachment sites, allowing for two distinct cellular signaling pathways in response to fluid shear stress, one transmitted by glycocalyx core proteins as a torque that acts on the actin cortical web (ACW) and DPAB, and the other emanating from focal adhesions and stress fibers at the basal and apical membranes of the cell.

Adipose tissue serves as a host reservoir for the protozoan Trypanosoma cruzi, the causative organism in Chagas disease. Gap junctions interconnect cells of most tissues, serving to synchronize cell activities including secretion in glandular tissue, and we have previously demonstrated that gap junctions are altered in various tissues and cells infected with T. cruzi. Herein, we examined the gap junction protein connexin 43 (Cx43) expression in infected adipose tissues. Adipose tissue is the largest endocrine organ of the body and is also involved in other physiological functions. In mammals, it is primarily composed of white adipocytes. Although gap junctions are a prominent feature of brown adipocytes, they have not been explored extensively in white adipocytes, especially in the setting of infection. Thus, we examined functional coupling in both white and brown adipocytes in mice. Injection of electrical current or the dye Lucifer Yellow into adipocytes within fat tissue spread to adjacent cells, which was reduced by treatment with agents known to block gap junctions. Moreover, Cx43 was detected in both brown and white fat tissue. At thirty and ninety days post-infection, Cx43 was downregulated in brown adipocytes and upregulated in white adipocytes. Gap junction-mediated intercellular communication likely contributes to hormone secretion and other functions in white adipose tissue and to nonshivering thermogenesis in brown fat, and modulation of the coupling by T. cruzi infection is expected to impact these functions.

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.

Osteocytes in the lacunar-canalicular system of the bone are thought to be the cells that sense mechanical loading and transduce mechanical strain into biomechanical responses. The goal of this study was to evaluate the extent to which focal mechanical stimulation of osteocyte cell body and process led to activation of the cells, and determine whether integrin attachments play a role in osteocyte activation. We use a novel Stokesian fluid stimulus probe to hydrodynamically load osteocyte processes vs. cell bodies in murine long bone osteocyte Y4 (MLO-Y4) cells with physiological-level forces

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.

Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm(2) at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF(164), play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.

Insulin-like growth factor I (IGF-I) is expressed in many tissues, including bone, and acts on the proliferation and differentiation of osteoblasts as an autocrine/paracrine regulator. Tight-junction proteins have been detected in osteoblasts, and direct cell-to-cell interactions may modulate osteoblast function with respect, for example, to gap junctions. In order to investigate the regulation of expression of tight-junction molecules and of function during bone differentiation, osteoblast-like MC3T3-E1 cells and osteocyte-like MLO-Y4 cells were treated with IGF-I. In both MC3T3-E1 cells and MLO-Y4 cells, the tight-junction molecules occludin, claudin-1, -2, and -6, and the gap-junction molecule connexin 43 (Cx43) were detected by reverse transcription with polymerase chain reaction. In MC3T3-E1 cells but not MLO-Y4 cells, mRNAs of claudin-1, -2, and -6, Cx43, and type I collagen, and proteins of claudin-1 and Cx43 were increased after treatment with IGF-I. Such treatment significantly decreased paracellular permeability in MC3T3-E1 cells. The expression of claudin-1 in MC3T3-E1 cells after IGF-I treatment was mainly upregulated via a mitogen-activated protein (MAP)-kinase pathway and, in part, modulated by a PI3-kinase pathway, whereas Cx43 expression and the mediated gap-junctional intercellular communication protein did not contribute to the upregulation. Furthermore, in MC3T3-E1 cells during wound healing, upregulation of claudin-1 was observed together with an increase of IGF-I and type I collagen. These findings suggest that the induction of tight-junction protein claudin-1 and paracellular permeability during the differentiation of osteoblast-like MC3T3-E1 cells after treatment with IGF-I is regulated via a MAP-kinase pathway, but not with respect to gap junctions.

Aquaporin-4 is a water channel predominantly found in astrocytes in the central nervous system and is believed to play a critical role in the formation and maintenance of the blood–brain barrier and in water secretion from the brain. As enteric glial cells were found to share several similarities with astrocytes, we hypothesized that enteric glia might also contain aquaporin-4. We used immunohistochemistry to identify aquaporin-4 in the myenteric and submucosal plexuses of the mouse and the rat colon. We found that sub-populations of neurons in both enteric plexuses were positively labeled for human aquaporin-4. Double staining of the enteric ganglia with antibodies to the neuronal marker neurofilament–heavy chain 100 and to aquaporin-4 showed that a minority of myenteric neurons were aquaporin-4 positive (about 12% in the mouse and 13% in the rat). In contrast, in the submucosal plexus significant numbers of neurons were positive for aquaporin-4 (about 79% in both the mouse and the rat). Double labeling for aquaporin-4 and for the glial marker glial fibrillary acidic protein verified that glial cells were not immunoreactive to aquaporin-4. We further confirmed our findings with additional aquaporin-4 antibodies and Western blot analysis. We found that, in addition to expressing aquaporin-4, the myenteric plexus and, to a greater extent, the submucosal plexus both expressed aquaporin-1. We conclude that neurons rather than glial cells contain aquaporin-4 in the colonic enteric plexuses. It is known that submucosal neurons control transport processes in the intestinal mucosa, and the high percentage of aquaporin-4-postive sub-mucosal neurons suggests that aquaporin-4 contributes to this function.

We tested the hypothesis that fluid shear stress (τ) modifies the expression, function, and distribution of junctional proteins [connexin (Cx)43, Cx45, and zona occludens (ZO)-1] in cultured bone cells. Cell lines with osteoblastic (MC3T3-E1 cells) and osteocytic (MLO-Y4 cells) phenotypes were exposed to τ-values of 5 or 20 dyn/cm2 for 1–3 h. Immunostaining indicated that at 5 dyn/cm2, the distribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cell membranes; at 20 dyn/cm2, disruption was more severe. Intercellular coupling was significantly decreased at both shear stress levels. Western blots showed the downregulation of membrane-bound Cx43 and ZO-1 and the upregulation of cytosolic Cx43 and Cx45 at different levels of shear stress. Similarly, Northern blots revealed that expression of Cx43, Cx45, and ZO-1 was selectively up- and downregulated in response to different shear stress levels. These results indicate that in cultured bone cells, fluid shear stress disrupts junctional communication, rearranges junctional proteins, and determines de novo synthesis of specific connexins to an extent that depends on the magnitude of the shear stress. Such disconnection from the bone cell network may provide part of the signal whereby the disconnected cells or the remaining network initiate focal bone remodeling.