Jean X. Jiang

ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels

J. Neurochem. (2011) 118, 826–840.

Amyloid β-Induced Death in Neurons Involves Glial and Neuronal Hemichannels

The mechanisms involved in Alzheimers disease are not completely understood and how glial cells contribute to this neurodegenerative disease remains to be elucidated. Because inflammatory treatments and products released from activated microglia increase glial hemichannel activity, we investigated whether amyloid-β peptide (Aβ) could regulate these channels in glial cells and affect neuronal viability. Microglia, astrocytes, or neuronal cultures as well as acute hippocampal slices made from GFAP-eGFP transgenic mice were treated with the active fragment of Aβ. Hemichannel activity was monitored by single-channel recordings and by time-lapse ethidium uptake, whereas neuronal death was assessed by Fluoro-Jade C staining. We report that low concentrations of Aβ25–35 increased hemichannel activity in all three cell types and microglia initiate these effects triggered by Aβ. Finally, neuronal damage occurs by activation of neuronal hemichannels induced by ATP and glutamate released from Aβ25–35-activated glia. These responses were observed in the presence of external calcium and were differently inhibited by hemichannel blockers, whereas the Aβ25–35-induced neuronal damage was importantly reduced in acute slices made from Cx43 knock-out mice. Thus, Aβ leads to a cascade of hemichannel activation in which microglia promote the release of glutamate and ATP through glial (microglia and astrocytes) hemichannels that induces neuronal death by triggering hemichannels in neurons. Consequently, this work opens novel avenues for alternative treatments that target glial cells and neurons to maintain neuronal survival in the presence of Aβ.

Effects of Mechanical Strain on the Function of Gap Junctions in Osteocytes Are Mediated through the Prostaglandin EP2 Receptor

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.

Expression of Functional Gap Junctions and Regulation by Fluid Flow in Osteocyte-Like MLO-Y4 Cells†

Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte‐like cell line MLO‐Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO‐Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO‐Y4 cells and approximately 5% of Cx43 protein is phosphorylated. MLO‐Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. “Scrape‐loading” dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte‐like cells was not observed in osteoblast‐like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte‐like MLO‐Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the cytoplasm also suggest that Cx43 may have unknown functions in addition to forming gap junctions. These studies indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.

PGE2 Is Essential for Gap Junction-Mediated Intercellular Communication between Osteocyte-Like MLO-Y4 Cells in Response to Mechanical Strain

We have observed, in our previous studies, that fluid flow increases gap junction-mediated intercellular coupling and the expression of a gap junction protein, connexin 43, in osteocyte-like MLO-Y4 cells. Interestingly, this stimulation is further enhanced during the poststress period, indicating that a released factor(s) is likely to be involved. Here, we report that the conditioned medium obtained from the fluid flow-treated MLO-Y4 cells increased the number of functional gap junctions and connexin 43 protein. These changes are similar to those observed in MLO-Y4 cells directly exposed to fluid flow. Fluid flow was found to induce PGE(2) release and increase cyclooxygenase 2 expression. Treatment of the cells with PGE(2) had the same effect as fluid flow, suggesting that PGE(2) could be responsible for these autocrine effects. When PGE(2) was depleted from the fluid flow-conditioned medium, the stimulatory effect on gap junctions was partially, but significantly, decreased. Addition of the cyclooxygenase inhibitor, indomethacin, partially blocked the stimulatory effects of mechanical strain on gap junctions. Taken together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated, in part, by the release of PGE(2). Hence, PGE(2) is an essential mediator between mechanical strain and gap junctions in osteocyte-like cells.

Mechanical stress-activated integrin α5β1 induces opening of connexin 43 hemichannels

The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5β1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC. Direct mechanical perturbation via magnetic beads or conformational activation of integrin α5β1 leads to the opening of the Cx43 HC, and this role of the integrin is independent of its association with an extracellular fibronectin substrate. PI3K signaling is responsible for the shear stress-induced conformational activation of integrin α5β1 leading to the opening of the HC. These results identify an unconventional function of integrin that acts as a mechanical tether to induce opening of the HC and provide a mechanism connecting the effect of mechanical forces directly to anabolic function of the bone.

Mechanical Induction of PGE2 in Osteocytes Blocks Glucocorticoid-Induced Apoptosis Through Both the β-Catenin and PKA Pathways

Glucocorticoids are known to induce osteocyte apoptosis, whereas mechanical loading has been shown to sustain osteocyte viability. Here we show that mechanical loading in the form of fluid‐flow shear stress blocks dexamethasone‐induced apoptosis of osteocyte‐like cells (MLO‐Y4). Prostaglandin E2 (PGE2), a rapidly induced signaling molecule produced by osteocytes, was shown to be protective against dexamethasone‐induced apoptosis, whereas indomethacin reversed the antiapoptotic effects of shear stress. This protective effect of shear stress was mediated through EP2 and EP4 receptors, leading to activation of the cAMP/protein kinase A signaling pathway. Activation of phosphatidylinositol 3‐kinase, an inhibitor of glycogen synthesis kinase 3, also occurred, leading to the nuclear translocation of β‐catenin, an important signal transducer of the Wnt signaling pathway. Both shear stress and prostaglandin increased the phosphorylation of glycogen synthesis kinase 3 α/β. Lithium chloride, an activator of the Wnt pathway, also was protective against glucocorticoid‐induced apoptosis. Whereas it is known that mechanical loading increases cyclooxygenase‐2 and EP2 receptor expression and prostaglandin production, dexamethasone was shown to inhibit expression of these components of the prostaglandin pathway and to reduce β‐catenin protein expression. β‐catenin siRNA knockdown experiments abrogated the protective effects of PGE2, confirming the central role of β‐catenin in mediating the protection against dexamethasone‐induced cell death. Our data support a central role for PGE2 acting through the cAMP/PKA and β‐catenin signaling pathways in the protection of osteocyte apoptosis by fluid‐flow shear stress.

Glucocorticoid-induced autophagy in osteocytes.

Glucocorticoid (GC) therapy is the most frequent cause of secondary osteoporosis. In this study we have demonstrated that GC treatment induced the development of autophagy, preserving osteocyte viability. GC treatment resulted in an increase in autophagy markers and the accumulation of autophagosome vacuoles in vitro and in vivo promoted the onset of the osteocyte autophagy, as determined by expression of autophagy markers in an animal model of GC‐induced osteoporosis. An autophagy inhibitor reversed the protective effects of GCs. The effects of GCs on osteocytes were in contrast to tumor necrosis factor α (TNF‐α), which induced apoptosis but not autophagy. Together this study reveals a novel mechanism for the effect of GC on osteocytes, shedding new insight into mechanisms responsible for bone loss in patients receiving GC therapy.

Biological Role of Connexin Intercellular Channels and Hemichannels

Gap junctions (GJ) and hemichannels (HC) formed from the protein subunits called connexins are transmembrane conduits for the exchange of small molecules and ions. Connexins and another group of HC-forming proteins, pannexins comprise the two families of transmembrane proteins ubiquitously distributed in vertebrates. Most cell types express more than one connexin or pannexin. While connexin expression and channel activity may vary as a function of physiological and pathological states of the cell and tissue, only a few studies suggest the involvement of pannexin HC in acquired pathological conditions. Importantly, genetic mutations in connexin appear to interfere with GJ and HC function which results in several diseases. Thus connexins could serve as potential drug target for therapeutic intervention. Growing evidence suggests that diseases resulting from HC dysfunction might open a new direction for development of specific HC reagents. This review provides a comprehensive overview of the current studies of GJ and HC formed by connexins and pannexins in various tissue and organ systems including heart, central nervous system, kidney, mammary glands, ovary, testis, lens, retina, inner ear, bone, cartilage, lung and liver. In addition, present knowledge of the role of GJ and HC in cell cycle progression, carcinogenesis and stem cell development is also discussed.

Adaptation of connexin 43-hemichannel prostaglandin release to mechanical loading

Bone tissues respond to mechanical loading/unloading regimens to accommodate (re)modeling requirements; however, the underlying molecular mechanism responsible for these responses is largely unknown. Previously, we reported that connexin (Cx) 43 hemichannels in mechanosensing osteocytes mediate the release of prostaglandin, PGE2, a crucial factor for bone formation in response to anabolic loading. We show here that the opening of hemichannels and release of PGE2 by shear stress were significantly inhibited by a potent antibody we developed that specifically blocks Cx43-hemichannels, but not gap junctions or other channels. The opening of hemichannels and release of PGE2 are magnitude-dependent on the level of shear stress. Insertion of a rest period between stress enhances this response. Hemichannels gradually close after 24 h of continuous shear stress corresponding with reduced Cx43 expression on the cell surface, thereby reducing any potential negative effects of channels staying open for extended periods. These data suggest that Cx43-hemichannel activity associated with PGE2 release is adaptively regulated by mechanical loading to provide an effective means of regulating levels of extracellular signaling molecules responsible for initiation of bone (re)modeling.