Manuel A. Riquelme

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.

Differential impact of adenosine nucleotides released by osteocytes on breast cancer growth and bone metastasis

Extracellular ATP has been shown to either inhibit or promote cancer growth and migration; however, the mechanism underlying this discrepancy remained elusive. Here we demonstrate the divergent roles of ATP and adenosine released by bone osteocytes on breast cancers. We showed that conditioned media (CM) collected from osteocytes treated with alendronate (AD), a bisphosphonate drug, inhibited the migration of human breast cancer MDA-MB-231 cells. Removal of the extracellular ATP by apyrase in CM abolished this effect, suggesting the involvement of ATP. ATP exerted its inhibitory effect through the activation of purinergic P2X receptor signaling in breast cancer cells evidenced by the attenuation of the inhibition by an antagonist, oxidized ATP, as well as knocking down P2X7 with small interfering RNA (siRNA), and the inhibition of migration by an agonist, BzATP. Intriguingly, ATP had a biphasic effect on breast cancer cells—lower dosage inhibited but higher dosage promoted its migration. The stimulatory effect on migration was blocked by an adenosine receptor antagonist, MRS1754, ARL67156, an ecto-ATPase inhibitor, and A2A receptor siRNA, suggesting that in contrast to ATP, adenosine, a metabolic product of ATP, promoted migration of breast cancer cells. Consistently, non-hydrolyzable ATP, ATPγS, only inhibited but did not promote cancer cell migration. ATP also had a similar inhibitory effect on the Py8119 mouse mammary carcinoma cells; however, adenosine had no effect owing to the absence of the A2A receptor. Consistently, ATPγS inhibited, whereas adenosine promoted anchorage-independent growth of MDA-MB-231 cells. Our in vivo xenograft study showed a significant delay of tumor growth with the treatment of ATPγS. Moreover, the extent of bone metastasis in a mouse intratibial model was significantly reduced with the treatment of ATPγS. Together, our results suggest the distinct roles of ATP and adenosine released by osteocytes and the activation of corresponding receptors P2X7 and A2A signaling on breast cancer cell growth, migration and bone metastasis.

Direct Regulation of Osteocytic Connexin 43 Hemichannels through AKT Kinase Activated by Mechanical Stimulation

Background: Opening of Cx43 hemichannels by mechanical stress releases factors important for bone remodeling; however, the regulatory mechanism is unknown. Result: Upon mechanical stimulation, AKT phosphorylates integrin α5 and Cx43, increases interaction, and opens hemichannels. Conclusion: Phosphorylation of Cx43 and α5 by AKT is critical for hemichannel opening. Significance: This is the first report demonstrating the functional importance of AKT in regulation of Cx43 hemichannels. Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation. Here, we show that mechanical loading through fluid flow shear stress (FFSS) increased the level of active AKT, a downstream effector of PI3K, which is correlated with the opening of hemichannels. Both Cx43 and integrin α5 are directly phosphorylated by AKT. Inhibition of AKT activation significantly reduced FFSS-induced opening of hemichannels and disrupted the interaction between Cx43 and integrin α5. Moreover, AKT phosphorylation on Cx43 and integrin α5 enhanced their interaction. In contrast to the C terminus of wild-type Cx43, overexpression of the C-terminal mutant containing S373A, a consensus site previously shown to be phosphorylated by AKT, failed to bind with α5 and hence could not inhibit hemichannel opening. Together, our results suggest that AKT activated by FFSS directly phosphorylates Cx43 and integrin α5, and Ser-373 of Cx43 plays a predominant role in mediating the interaction between these two proteins and Cx43 hemichannel opening, a crucial step to mediate the anabolic function of mechanical loading in the bone.

Connexin 43 Channels Are Essential for Normal Bone Structure and Osteocyte Viability

Connexin (Cx) 43 serves important roles in bone function and development. Targeted deletion of Cx43 in osteoblasts or osteocytes leads to increased osteocyte apoptosis, osteoclast recruitment, and reduced biomechanical properties. Cx43 forms both gap junction channels and hemichannels, which mediate the communication between adjacent cells or between cell and extracellular environments, respectively. Two transgenic mouse models driven by a DMP1 promoter with the overexpression of dominant negative Cx43 mutants were generated to dissect the functional contribution of Cx43 gap junction channels and hemichannels in osteocytes. The R76W mutant blocks the gap junction channel, but not the hemichannel function, and the Δ130‐136 mutant inhibits activity of both types of channels. Δ130‐136 mice showed a significant increase in bone mineral density compared to wild‐type (WT) and R76W mice. Micro–computed tomography (µCT) analyses revealed a significant increase in total tissue and bone area in midshaft cortical bone of Δ130‐136 mice. The bone marrow cavity was expanded, whereas the cortical thickness was increased and associated with increased bone formation along the periosteal area. However, there is no significant alteration in the structure of trabecular bone. Histologic sections of the midshaft showed increased apoptotic osteocytes in Δ130‐136, but not in WT and R76W, mice which correlated with altered biomechanical and estimated bone material properties. Osteoclasts were increased along the endocortical surface in both transgenic mice with a greater effect in Δ130‐136 mice that likely contributed to the increased marrow cavity. Interestingly, the overall expression of serum bone formation and resorption markers were higher in R76W mice. These findings suggest that osteocytic Cx43 channels play distinctive roles in the bone; hemichannels play a dominant role in regulating osteocyte survival, endocortical bone resorption, and periosteal apposition, and gap junction communication is involved in the process of bone remodeling.

Connexin 43 Channels Protect Osteocytes Against Oxidative Stress–Induced Cell Death

The increased osteocyte death by oxidative stress (OS) during aging is a major cause contributing to the impairment of bone quality and bone loss. However, the underlying molecular mechanism is largely unknown. Here, we show that H2O2 induced cell death of primary osteocytes and osteocytic MLO‐Y4 cells, and also caused dose‐dependent decreased expression of gap junction and hemichannel‐forming connexin 43 (Cx43). The decrease of Cx43 expression was also demonstrated with the treatment of other oxidants, rotenone and menadione. Antioxidant reversed the effects of oxidants on Cx43 expression and osteocyte cell death. Cx43 protein was also much lower in the osteocytes from 20‐month‐old as opposed to the 5‐week‐old or 20‐week old mice. Dye transfer assay showed that H2O2 reduced the gap junction intercellular communication (GJIC). In contrast to the effect on GJIC, there was a dose‐dependent increase of hemichannel function by H2O2, which was correlated with the increased cell surface expression of Cx43. Cx43(E2) antibody, an antibody that specifically blocks Cx43 hemichannel activity but not gap junctions, completely blocked dye uptake induced by H2O2 and further exacerbated H2O2‐induced osteocytic cell death. In addition, knockdown of Cx43 expression by small interfering RNA (siRNA) increased the susceptibility of the cells to OS‐induced death. Together, our study provides a novel cell protective mechanism mediated by osteocytic Cx43 channels against OS.

Cataract-Causing Mutation of Human Connexin 46 Impairs Gap Junction, but Increases Hemichannel Function and Cell Death

Connexin channels play a critical role in maintaining metabolic homeostasis and transparency of the lens. Mutations in connexin genes are linked to congenital cataracts in humans. The G143R missense mutation on connexin (Cx) 46 was recently reported to be associated with congenital Coppock cataracts. Here, we showed that the G143R mutation decreased Cx46 gap junctional coupling in a dominant negative manner; however, it significantly increased gap junctional plaques. The G143R mutant also increased hemichannel activity, inversely correlated with the level of Cx46 protein on the cell surface. The interaction between cytoplasmic loop domain and C-terminus has been shown to be involved in gating of connexin channels. Interestingly, the G143R mutation enhanced the interaction between intracellular loop and Cx46. Furthermore, this mutation decreased cell viability and the resistance of the cells to oxidative stress, primarily due to the increased hemichannel function. Together, these results suggest that mutation of this highly conserved residue on the cytoplasmic loop domain of Cx46 enhances its interaction with the C-terminus, resulting in a reduction of gap junction channel function, but increased hemichannel function. This combination leads to the development of human congenital cataracts.

Osteocytic connexin hemichannels suppress breast cancer growth and bone metastasis

Although the skeleton is one of the predominant sites for breast cancer metastasis, why breast cancer cells often become dormant after homing to bone is not well understood. Here, we reported an intrinsic self-defense mechanism of bone cells against breast cancer cells: a critical role of connexin (Cx) 43 hemichannels in osteocytes in the suppression of breast cancer bone metastasis. Cx43 hemichannels allow passage of small molecules between the intracellular and extracellular environments. The treatment of bisphosphonate drugs, either alendronate (ALN) or zoledronic acid (ZOL), opened Cx43 hemichannels in osteocytes. Conditioned media (CM) collected from MLO-Y4 osteocyte cells treated with bisphosphonates inhibited the anchorage-independent growth, migration and invasion of MDA-MB-231 human breast cancer cells and Py8119 mouse mammary carcinoma cells, and this inhibitory effect was attenuated with Cx43(E2), a specific hemichannel-blocking antibody. The opening of osteocytic Cx43 hemichannels by mechanical stimulation had similar inhibitory effects on breast cancer cells and this inhibition was attenuated by Cx43(E2) antibody as well. These inhibitory effects on cancer cells were mediated by ATP released from osteocyte Cx43 hemichannels. Furthermore, both Cx43 osteocyte-specific knockout mice and osteocyte-specific Δ130-136 transgenic mice with impaired Cx43 gap junctions and hemichannels showed significantly increased tumor growth and attenuated the inhibitory effect of ZOL. However, R76W transgenic mice with functional hemichannels but not gap junctions in osteocytes did not display a significant difference. Together, our studies establish the specific inhibitory role of osteocytic Cx43 hemichannels, and exploiting the activity of this channel could serve as a de novo therapeutic strategy.

14-3-3θ Facilitates plasma membrane delivery and function of mechanosensitive connexin 43 hemichannels

ABSTRACT Intracellular signaling in osteocytes activated by mechanical loading is important for bone formation and remodeling. These signaling events are mediated by small modulators released from Cx43 hemichannels (HC). We have recently shown that integrin &agr;5 senses the mechanical stimulation and induces the opening of Cx43 HC; however, the underlying mechanism is unknown. Here, we show that both Cx43 and integrin &agr;5 interact with 14-3-3&thgr;, and this interaction is required for the opening of Cx43 HC upon mechanical stress. The absence of 14-3-3&thgr; prevented the interaction between Cx43 and integrin &agr;5, and blocked HC opening. Furthermore, it decreased the transport of Cx43 and integrin &agr;5 from the Golgi apparatus to the plasma membrane. Mechanical loading promoted the movement of Cx43 to the surface which was associated not only with an increase in 14-3-3&thgr; levels but also its interaction with Cx43 and integrin &agr;5. This stimulatory effect on forward transport by mechanical loading was attenuated in the absence of 14-3-3&thgr; and the majority of the Cx43 accumulated in the Golgi. Disruption of the Golgi by brefeldin A reduced the association of Cx43 and integrin &agr;5 with 14-3-3&thgr;, further suggesting that the interaction is likely to occur in the Golgi. Together, these results define a previously unidentified, scaffolding role of 14-3-3&thgr; in assisting the delivery of Cx43 and integrin &agr;5 to the plasma membrane for the formation of mechanosensitive HC in osteocytes.

Mitogen-activated Protein Kinase (MAPK) Activated by Prostaglandin E2 Phosphorylates Connexin 43 and Closes Osteocytic Hemichannels in Response to Continuous Flow Shear Stress.

Background: Sustained Cx43 hemichannel opening is detrimental to bone cells; however, the mechanism underlying the closure of hemichannels was unknown. Results: Extracellular prostaglandin E2 released by hemichannels activates MAPK, leading to Cx43 phosphorylation and hemichannel closure. Conclusion: Osteocytic Cx43 hemichannels is regulated by a feedback inhibition mechanism. Significance: This study uncovers a novel pathway in fine-tuning Cx43 hemichannels in response to mechanical stimulation. Cx43 hemichannels serve as a portal for the release of prostaglandins, a critical process in mediating biological responses of mechanical loading on bone formation and remodeling. We have previously observed that fluid flow shear stress (FFSS) opens hemichannels; however, sustained FFSS results in hemichannel closure, as continuous opening of hemichannels is detrimental to cell viability and bone remodeling. However, the mechanism that regulates the closure of the hemichannels is unknown. Here, we show that activation of p44/42 ERK upon continuous FFSS leads to Cx43 phosphorylation at Ser279-Ser282, sites known to be phosphorylated sites by p44/42 MAPK. Incubation of osteocytic MLO-Y4 cells with conditioned media (CM) collected after continuous FFSS increased MAPK-dependent phosphorylation of Cx43. CM treatment inhibited hemichannel opening and this inhibition was reversed when cells were pretreated with the MAPK pathway inhibitor. We found that prostaglandin E2 (PGE2) accumulates in the CM in a time-dependent manner. Treatment with PGE2 increased phospho-p44/42 ERK levels and also Cx43 phosphorylation at Ser279-Ser282 sites. Depletion of PGE2 from CM, and pre-treatment with a p44/42 ERK pathway-specific inhibitor, resulted in a complete inhibition of ERK-dependent Cx43 phosphorylation and attenuated the inhibition of hemichannels by CM and PGE2. Consistently, the opening of hemichannels by FFSS was blocked by PGE2 and CM and this blockage was reversed by U0126 and the CM depleted of PGE2. A similar observation was also obtained in isolated primary osteocytes. Together, results from this study suggest that extracellular PGE2 accumulated after continuous FFSS is responsible for activation of p44/42 ERK signaling and subsequently, direct Cx43 phosphorylation by activated ERK leads to hemichannel closure.

Pannexin 1 channels in skeletal muscles

Normal myotubes and adult innervated skeletal myofibers express the glycoprotein pannexin1 (Panx1). Six of them form a “gap junction hemichannel-like” structure that connects the cytoplasm with the extracellular space; here they will be called Panx1 channels. These are poorly selective channels permeable to ions, small metabolic substrate, and signaling molecules. So far little is known about the role of Panx1 channels in muscles but skeletal muscles of Panx1−/− mice do not show an evident phenotype. Innervated adult fast and slow skeletal myofibers show Panx1 reactivity in close proximity to dihydropyridine receptors in the sarcolemma of T-tubules. These Panx1 channels are activated by electrical stimulation and extracellular ATP. Panx1 channels play a relevant role in potentiation of muscle contraction because they allow release of ATP and uptake of glucose, two molecules required for this response. In support of this notion, the absence of Panx1 abrogates the potentiation of muscle contraction elicited by repetitive electrical stimulation, which is reversed by exogenously applied ATP. Phosphorylation of Panx1 Thr and Ser residues might be involved in Panx1 channel activation since it is enhanced during potentiation of muscle contraction. Under denervation, Panx1 levels are upregulated and this partially explains the reduction in electrochemical gradient, however its absence does not prevent denervation-induced atrophy but prevents the higher oxidative state. Panx1 also forms functional channels at the cell surface of myotubes and their functional state has been associated with intracellular Ca2+ signals and regulation of myotube plasticity evoked by electrical stimulation. We proposed that Panx1 channels participate as ATP channels and help to keep a normal oxidative state in skeletal muscles.