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 dyne͞cm 2 ; 1 dyne ‫؍‬ 10 N). 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. mechanotransduction ͉ actin cortical web ͉ dense peripheral actin band H emodynamic shearing stresses on endothelial cells (ECs) are widely recognized as playing a vital role in the regulation of vessel wall remodeling, cellular signaling, mass transport, red and white cell interaction, and atherogenesis (1-3). The possible roles of the endothelial glycocalyx (EG) in this regulation as a molecular sieve, as a barrier and modulator of interactions between blood cells and ECs, and as a mechanotransducer of fluid shear stress have been studied more recently (4-7). Relatively little is known about the specific proteins in the EG, although hyaluronan, chondroitin, and heparan sulfate play a significant role in its assembly (8, 9). In the early 1990s, investigators first observed that the shear-induced dilation of small arteries was abolished when sialic acids were removed from the EG by neuraminidase (10). Florian et al. (11) recently verified the presence of heparan sulfate proteoglycan (HSPG) in the glycocalyx of cultured bovine aortic ECs and demonstrated that partial removal of HSPG with heparinase completely blocked shear-induced NO release. A puzzling and still not understood consequence of EG degradation was the observation that shear-induced NO production was greatly inhibited without apparent effect on shear-dependent vasodilation due to prostaglandin I 2 release (12). Squire et al. (13) showed that the ultrastructural organization of the EG was quasiperiodic, anchored to a geodesic-like scaffold of hexagonally arranged filamentous actin (F-actin) filaments forming an actin cortical web (ACW) (14) just beneath the plasmalemma. A fundamental question addressed in ref. 7 is how fluid shear stresses acting at the surface of the EG are transmitted to this ACW if there is essentially ...

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.

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 physiologicallevel forces <10 pN without probe contact, and measured intracellular Ca 2+ responses. Our results indicate that osteocyte processes are extremely responsive to piconewton-level mechanical loading, whereas the osteocyte cell body and processes with no local attachment sites are not. Ca 2+ signals generated at stimulated sites spread within the processes with average velocity of 5.6 μm/s. Using the near-infrared fluorescence probe IntegriSense 750, we demonstrated that inhibition of α V β 3 integrin attachment sites compromises the response to probe stimulation. Moreover, using apyrase, an extracellular ATP scavenger, we showed that Ca 2+ signaling from the osteocyte process to the cell body was greatly diminished, and thus dependent on ATP-mediated autocrine signaling. These findings are consistent with the hypothesis that osteocytes in situ are highly polarized cells, where mechanotransduction occurs at substrate attachment sites along the processes at force levels predicted to occur at integrin attachment sites in vivo. We also demonstrate the essential role of α V β 3 integrin in osteocyte-polarized mechanosensing and mechanotransduction. intracellular calcium | cell process attachment | fluid flow activation | purinergic signaling A dynamic and complex structure such as bone has the ability to adapt and to adjust to changes in its functional environment. Emerging evidence suggests that osteocytes play an important role in regulating bone mechanoadaptation and in adjusting and maintaining bone mass (1-6). Osteocytes, which account for ∼90% of the entire bone cell population, are viewed as the main mechanosensing cells that detect whole tissue mechanical loading due to their unique distribution throughout the mineralized matrix and to their connection to the neighboring osteocytes and osteoblasts via gap junctions (7).To sense and respond to mechanical loading and thus maintain bone homeostasis, the osteocyte must be properly anchored to its extracellular surroundings (reviewed in ref. 8). In vivo, the osteocyte cell processes in the lacunar-canalicular system (LCS) are surrounded by transverse tethering elements (9-11) and are directly connected to the canalicular wall at discrete attachment sites (12, 13) that contain β 3 integrin (12). Integrins are transmembrane proteins that link the cell's cytoskeleton to the extracellular matrix and are recognized for their key roles in mechanosensory transduction (14, 15). All bone cells express...

2 for 1-3 h. Immunostaining indicated that at 5 dyn/cm 2 , the distribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cell membranes; at 20 dyn/cm 2 , 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. gap junctions; connexin; zona occludens-1; mechanotransduction; bone remodeling BONE DRAMATICALLY CHANGES its structure and mass in response to static and dynamic loads. It has been well established that bending loads in bone cause small deformations in the bone matrix, which in turn generate fluid pressure differences that lead to fluid flow from the compression to the tension side. The resulting fluid shear stress () is proposed to be one of the factors by which the bone cell network senses mechanical loading (7,51,54,61). At physiological whole-tissue strain levels (Ͻ0.2%), culture studies have demonstrated that fluid flow is a more potent stimulator of bone cells than is substrate deformation itself (38, 59).

Osteocyte apoptosis is required to induce intracortical bone remodeling following 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 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, 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 P2X7R in ATP release; thus we examined P2X7R-deficient mice and WT mice treated with P2X7R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find-me signal. P2X7RKO 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 P2X7RKO 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 percent. These results indicate that activation of Panx1 and P2X7R 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.

Type 1 diabetes (T1D) causes a range of skeletal problems, including reduced bone density and increased risk for bone fractures. However, mechanisms underlying skeletal complications in diabetes are still not well understood. We hypothesize that high glucose levels in T1D alters expression and function of purinergic receptors (P2Rs) and pannexin 1 (Panx1) channels, and thereby impairs ATP signaling that is essential for proper bone response to mechanical loading and maintenance of skeletal integrity. We first established a key role for P2X7 receptor-Panx1 in osteocyte mechanosignaling by showing that these proteins are co-expressed to provide a major pathway for flow-induced ATP release. To simulate in vitro the glucose levels to which bone cells are exposed in healthy vs. diabetic bones, we cultured osteoblast and osteocyte cell lines for 10 days in medium containing 5.5 or 25 mM glucose. High glucose effects on expression and function of P2Rs and Panx1 channels were determined by Western Blot analysis, quantification of Ca2+ responses to P2R agonists and oscillatory fluid shear stress (± 10 dyne/cm2), and measurement of flow-induced ATP release. Diabetic C57BL/6J-Ins2Akita mice were used to evaluate in vivo effects of high glucose on P2R and Panx1. Western blotting indicated altered P2X7R, P2Y2R and P2Y4R expression in high glucose exposed bone cells, and in diabetic bone tissue. Moreover, high glucose blunted normal P2R- and flow-induced Ca2+ signaling and ATP release from osteocytes. These findings indicate that T1D impairs load-induced ATP signaling in osteocytes and affects osteoblast function, which are essential for maintaining bone health.

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.

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.