Sumin Gu

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.

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.

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.

Identification and characterization of an amino acid transporter expressed differentially in liver.

Cellular metabolic needs are fulfilled by transport of amino acids across the plasma membrane by means of specialized transporter proteins. Although many of the classical amino acid transporters have been characterized functionally, less than half of these proteins have been cloned. In this report, we identify and characterize a cDNA encoding a plasma membrane amino acid transporter. The deduced amino acid sequence is 505 residues and is highly hydrophobic with the likely predicted structure of 9 transmembrane domains, which putatively place the amino terminus in the cytoplasm and the carboxy terminus on the cell surface. Expression of the cRNA in Xenopus laevis oocytes revealed strong transport activities specific for histidine and glutamine. This protein is a Na(+)- and pH-dependent transporter and tolerates substitution of Na(+) by Li(+). Furthermore, this transporter is not an obligatory exchanger because efflux occurs in the absence of influx. This transporter is expressed predominantly in the liver, although it is also present in the kidney, brain, and heart. In the liver, it is located in the plasma membrane of hepatocytes, and the strongest expression was detected in those adjacent to the central vein, gradually decreasing towards the portal tract. Because this protein displays functional similarities to the N-system amino acid transport, we have termed it mNAT, for murine N-system amino acid transporter. This is the first transporter gene identified within the N-system, one of the major amino acid transport systems in the body. The expression pattern displayed by mNAT suggests a potential role in hepatocyte physiology.

Tubby-like protein-1 mutations in autosomal recessive retinitis pigmentosa.

We suggest that antimony is able to enter the body from environmental sources other than cot mattresses and their covers. We determined antimony concentrations in babies with SIDS; stillborn babies; babies who died aged less than a week; and babies who died aged under 1 year. Samples of liver were collected at necropsy at the Hammersmith Hospital, London. Laboratory analysis was performed blind to group, and the code was not broken till all chemical analyses had been completed. Hepatic antimony concentration was determined in triplicate using hydridegeneration Atomic Absorption Spectroscopy (AAS). We took precautions to avoid any possible contamination of samples with antimony. There was a wide variation in hepatic antimony concentrations within the group, particularly in the oldest control group where there was one infant with a hepatic antimony concentration of 98 ng/g. Despite these wide fluctuations there were no statistically significant differences between antimony concentrations in the four groups (see table). These results do not support Richardson’s theory that antimony exposure is a cause of SIDS. However, it is apparent that there is evidence of both prenatal and postnatal exposures to antimony. Furthermore, the presence of antimony in the liver of stillborn babies suggests that antimony was accumulating during fetal life. Antimony from industrial sources is an environmental pollutant that is present in the food chain and in the atmosphere. It accumulates in the body, particularly in the skeleton. 5 It is possible that during pregnancy and lactation when enhanced bone remodelling of the maternal skeleton occurs, antimony may be released into maternal blood, and will thus be free to cross the placenta and accumulate in fetal tissues.

Characterization of an N-system amino acid transporter expressed in retina and its involvement in glutamine transport

We report here on the characterization of a mouse N-system amino acid transporter protein, which is involved in the transport of glutamine. This protein of 485 amino acids shares 52% sequence homology with an N-system amino acid transporter, mouse N-system amino acid transporter (mNAT) and its orthologs. Because this protein shares a high degree of sequence homology and functional similarity to mNAT, we named it mNAT2. mNAT2 is predominately expressed in the retina and to a slightly lesser extent in the brain. In the retina, it is located in the axons of ganglion cells in the nerve fiber layer and in the bundles of the optic nerve. Functional analysis of mNAT2 expressed in Xenopus oocytes revealed that the strongest transport activities were specific forl-glutamine. In addition, mNAT2 is a Na+- and pH-dependent, high affinity transporter and partially tolerates substitution of Na+ by Li+. Additionally, mNAT2 functions as a carrier-mediated transporter that facilitates efflux. The unique expression pattern and selective glutamine transport properties of mNAT2 suggest that it plays a specific role in the uptake of glutamine involved in the generation of the neurotransmitter glutamate in retina.

Aquaporin 0 enhances gap junction coupling via its cell adhesion function and interaction with connexin 50

Both connexin 50 (Cx50) and aquaporin 0 (AQP0) have important roles in lens development and homeostasis, and their mutations are associated with human congenital cataracts. We have previously shown that Cx50 directly interacts with AQP0. Here, we demonstrate the importance of the Cx50 intracellular loop (IL) domain in mediating the interaction with AQP0 in the lens in vivo. AQP0 significantly increased (~20–30%) the intercellular coupling and conductance of Cx50 gap junctions. However, this increase was not observed when the IL domain was replaced with those from other lens connexins. The Cx50–AQP0 interaction had no effect on Cx50 hemichannel function. A fusion protein containing three extracellular loop domains of AQP0 efficiently blocked the cell-to-cell adhesion of AQP0 and attenuated the stimulatory effect of AQP0 on Cx50 gap junction conductance. These data suggest that the specific interaction between Cx50 and AQP0 enhances the coupling of Cx50 gap junctions, but not hemichannels, through the cell adhesion function of AQP0. This result establishes a physiological role of AQP0 in the functional regulation of gap junction channels.

Mechanical Loading Stimulates Expression of Connexin 43 in Alveolar Bone Cells in the Tooth Movement Model

Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption.

Phosphorylation of Connexin 50 by Protein Kinase A Enhances Gap Junction and Hemichannel Function

Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ∼200-pS open state while simultaneously reducing transitions between this open state and a ∼65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ∼200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.

Autosomal recessive retinitis pigmentosa locus RP28 maps between D2S1337 and D2S286 on chromosome 2p11-p15 in an Indian family

Retinitis pigmentosa (RP) is a group of clinically and genetically heterogeneous disorders characterised by night blindness, constriction of visual field, and dystrophic changes of the retina. Previous genetic studies have shown extensive allelic and non-allelic genetic heterogeneity of RP. Here we describe an Indian family with multiple consanguineous marriages and a total of four patients with autosomal recessive (AR) RP. The homozygosity mapping strategy was successfully used and indicated close linkage between the disease locus and D2S380, D2S441, D2S291, and D2S1394 with maximum lod scores between 1.51-3.07 at θ=0.00. The analysis of multiply informative meioses maps the locus (RP28) for ARRP in this family between D1S1337 and D2S286 on 2p11-p15. The involvement of visinin (VSNL1), a promising candidate gene assigned to chromosome 2p by previous studies, has been excluded by the absence of linkage.