AJP: Cell Physiology current issue

Novel epigenetic regulation of skeletal muscle myosin heavy chain genes. Focus on "Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading"

Zwetsloot, K. A., Laye, M. J., Booth, F. W. — 2009-07-01

Multifunctional angiogenic factors: add GnRH to the list. Focus on "Gonadotropin-releasing hormone-regulated chemokine expression in human placentation"

Pearce, W. J. — 2009-07-01

Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading

Pandorf, C. E., Haddad, F., Wright, C., Bodell, P. W., Baldwin, K. M. — 2009-07-01

Recent advances in chromatin biology have enhanced our understanding of gene regulation. It is now widely appreciated that gene regulation is dependent upon post-translational modifications to the histones which package genes in the nucleus of cells. Active genes are known to be associated with acetylation of histones (H3ac) and trimethylation of lysine 4 in histone H3 (H3K4me3). Using chromatin immunoprecipitation (ChIP), we examined histone modifications at the myosin heavy chain (MHC) genes expressed in fast vs. slow fiber-type skeletal muscle, and in a model of muscle unloading, which results in a shift to fast MHC gene expression in slow muscles. Both H3ac and H3K4me3 varied directly with the transcriptional activity of the MHC genes in fast fiber-type plantaris and slow fiber-type soleus. During MHC transitions with muscle unloading, histone H3 at the type I MHC becomes de-acetylated in correspondence with down-regulation of that gene, while upregulation of the fast type IIx and IIb MHCs occurs in conjunction with enhanced H3ac in those MHCs. Enrichment of H3K4me3 is also increased at the type IIx and IIb MHCs when these genes are induced with muscle unloading. Downregulation of IIa MHC, however, was not associated with corresponding loss of H3ac or H3K4me3. These observations demonstrate the feasibility of using the ChIP assay to understand the native chromatin environment in adult skeletal muscle, and also suggest that the transcriptional state of types I, IIx and IIb MHC genes are sensitive to histone modifications both in different muscle fiber-types and in response to altered loading states.

Gonadotropin-releasing hormone-regulated chemokine expression in human placentation

2009-07-01

Placental expression of gonadotropin-releasing hormone (GnRH)-I and II, as well as their cognate receptor, coincides with a period of extensive remodeling of the maternal-fetal interface, near the end of the first trimester of pregnancy. To further define the role of GnRH in human placentation, we performed a microarray screen of HTR-8/SVneo trophoblasts to identify GnRH-regulated genes and their roles in placentation. This screen revealed that GnRH regulates the expression of four angiogenic chemokines: CXCL2, CXCL3, CXCL6, and CXCL8. The microarray data were subsequently confirmed by an extensive Q-PCR time-course analysis. CXCL8, a representative chemokine, was selected for further analysis and shown to be strongly expressed by trophoblasts at the maternal-fetal interface of the human placenta, as well as to accumulate in a GnRH-dependent manner in trophoblast-conditioned media in culture. Trophoblasts were subsequently shown to recruit lymphocytes (Jurkat T cells and primary peripheral blood T and uterine natural killer cells) in chemotaxis assays and this was shown to be GnRH dependent. Furthermore, this recruitment was shown to occur via the release of CXCR1/CXCR2 interacting chemokines, such as the CXCLs investigated in this study. This novel regulation of chemokines by GnRH signaling demonstrates the role of GnRH in regulating the recruitment of lymphocytes to the decidua and the possibility of a direct effect on spiral artery remodeling via the release of proangiogenic chemokines and secondary effects via release of angiogenic factors by recruited lymphocytes.

Pharmacological properties of a pore induced by raising intracellular Ca2+

2009-07-01

Recent studies on the P2X₇ receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca²⁺ concentration induces a pore opening similar to P2X₇ receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca²⁺ concentration is associated to P2X₇ receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X₇ receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5–10 µM) increased intracellular free Ca²⁺ concentration and induced pore formation with conductance of 421 ± 14 pS, half-time (t_1/2) for ethidium bromide uptake of 118 ± 17 s, and t_1/2 for Lucifer yellow of 122 ± 11 s. P2X₇ receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular Ca²⁺ concentration. However, 5-(N,N-hexamethylene)-amiloride, a P2X₇ receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular Ca²⁺ at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca⁺² induces a novel membrane pore pharmacologically different from the P2X₇ associated pore and hemigap-junction pore.

Simultaneous overexpression of Oct4 and Nanog abrogates terminal myogenesis

2009-07-01

Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.

MAGI-1 interacts with Slo1 channel proteins and suppresses Slo1 expression on the cell surface

Ridgway, L. D., Kim, E. Y., Dryer, S. E. — 2009-07-01

Large conductance Ca²⁺-activated K⁺ (BK_Ca) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BK_Ca channels using a bait construct derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues, including podocytes and the brain. The interaction between MAGI-1 and BK_Ca channels was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays in differentiated cells of a podocyte cell line and in human embryonic kidneys (HEK)293T cells transiently coexpressing MAGI-1a and three different COOH-terminal Slo1 variants. Coexpression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knockdown of endogenous MAGI-1 expression by small interfering RNA (siRNA) in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas overexpression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BK_Ca channels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BK_Ca channels in the kidney and possibly in other tissues.

Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1

Unal, E. S., Zhao, R., Goldman, I. D. — 2009-07-01

The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [³H]methotrexate (MTX) influx V_max; the MTX influx K_t was identical to that of wild-type (WT)-PCFT (1.5 µM). Consistent with the intrinsic functionality of E185A-PCFT, [³H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (~38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.

Tamoxifen inhibits BK channels in chick cochlea without alterations in voltage-dependent activation

Tong, M., Duncan, R. K. — 2009-07-01

Large-conductance, Ca²⁺-activated, and voltage-gated potassium channels (BK, BK_Ca, or Maxi-K) play an important role in electrical tuning in nonmammalian vertebrate hair cells. Systematic changes in tuning frequency along the tonotopic axis largely result from variations in BK channel kinetics, but the molecular changes underpinning these functional variations remain unknown. Auxiliary β₁ have been implicated in low-frequency tuning at the cochlear apex because these subunits dramatically slow channel kinetics. Tamoxifen (Tx), a (xeno)estrogen compound known to activate BK channels through the β-subunit, was used to test for the functional presence of β₁. The hypotheses were that Tx would activate the majority of BK channels in hair cells from the cochlear apex due to the presence of β₁ and that the level of activation would exhibit a tonotopic gradient following the expression profile of β₁. Outside-out patches of BK channels were excised from tall hair cells along the apical half of the chicken basilar papilla. In low-density patches, single-channel conductance was reduced and the averaged open probability was unaffected by Tx. In high-density patches, the amplitude of ensemble-averaged BK current was inhibited, whereas half-activation potential and activation kinetics were unaffected by Tx. In both cases, no tonotopic Tx-dependent activation of channel activity was observed. Therefore, contrary to the hypotheses, electrophysiological assessment suggests that molecular mechanisms other than auxiliary β-subunits are involved in generating a tonotopic distribution of BK channel kinetics and electric tuning in chick basilar papilla.

Endofacial competitive inhibition of the glucose transporter 1 activity by gossypol

2009-07-01

Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a K_i value of 20 µM. Likewise, GLUT1 fluorescence was quenched ~80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of d-glucose. Gossypol did not alter the affinity of d-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.

AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR

2009-07-01

The metabolic sensor AMP-activated protein kinase (AMPK) has emerged as an important link between cellular metabolic status and ion transport activity. We previously found that AMPK binds to and phosphorylates CFTR in vitro and inhibits PKA-dependent stimulation of CFTR channel gating in Calu-3 bronchial serous gland epithelial cells. To further characterize the mechanism of AMPK-dependent regulation of CFTR, whole cell patch-clamp measurements were performed with PKA activation in Calu-3 cells expressing either constitutively active or dominant-negative AMPK mutants (AMPK-CA or AMPK-DN). Baseline CFTR conductance in cells expressing AMPK-DN was substantially greater than controls, suggesting that tonic AMPK activity in these cells inhibits CFTR under basal conditions. Although baseline CFTR conductance in cells expressing AMPK-CA was comparable to that of controls, PKA stimulation of CFTR was completely blocked in AMPK-CA-expressing cells, suggesting that AMPK activation renders CFTR resistant to PKA activation in vivo. Phosphorylation studies of CFTR in human embryonic kidney-293 cells using tetracycline-inducible expression of AMPK-DN demonstrated AMPK-dependent phosphorylation of CFTR in vivo. However, AMPK activity modulation had no effect on CFTR in vivo phosphorylation in response to graded doses of PKA or PKC agonists. Thus, AMPK-dependent CFTR phosphorylation renders the channel resistant to activation by PKA and PKC without preventing phosphorylation by these kinases. We found that Ser768, a CFTR R domain residue considered to be an inhibitory PKA site, is the dominant site of AMPK phosphorylation in vitro. Ser-to-Ala mutation at this site enhanced baseline CFTR activity and rendered CFTR resistant to inhibition by AMPK, suggesting that AMPK phosphorylation at Ser768 is required for its inhibition of CFTR. In summary, our findings indicate that AMPK-dependent phosphorylation of CFTR inhibits CFTR activation by PKA, thereby tuning the PKA-responsiveness of CFTR to metabolic and other stresses in the cell.

Histamine hyperpolarizes human glioblastoma cells by activating the intermediate-conductance Ca2+-activated K+ channel

2009-07-01

The effects of histamine on the membrane potential and currents of human glioblastoma (GL-15) cells were investigated. In perforated whole cell configuration, short (3 s) applications of histamine (100 µM) hyperpolarized the membrane by activating a K⁺-selective current. The response involved the activation of the pyrilamine-sensitive H₁ receptor and Ca²⁺ release from thapsigargin-sensitive intracellular stores. The histamine-activated current was insensitive to tetraethylammonium (3 mM), iberiotoxin (100 nM), and d-tubocurarine (100 µM) but was markedly inhibited by charybdotoxin (100 nM), clotrimazole (1 µM), and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34, 1 µM), a pharmacological profile congruent with the intermediate conductance Ca²⁺-activated K⁺ (IK_Ca) channel. Cell-attached recordings confirmed that histamine activated a K⁺ channel with properties congruent with the IK_Ca channel (voltage independence, 22 pS unitary conductance and slight inward rectification in symmetrical 140 mM K⁺). More prolonged histamine applications (2–3 min) often evoked a sustained IK_Ca channel activity, which depended on a La²⁺ (10 µM)-sensitive Ca²⁺ influx. Intracellular Ca²⁺ measurements revealed that the sustained IK_Ca channel activity enhanced the histamine-induced Ca²⁺ signal, most likely by a hyperpolarization-induced increase in the driving force for Ca²⁺ influx. In virtually all cells examined we also observed the expression of the large conductance Ca²⁺-activated K⁺ (BK_Ca) channel, with a unitary conductance of ca. 230 pS in symmetrical 140 mM K⁺, and a Ca²⁺ dissociation constant [K_D(Ca)] of ca. 3 µM, at –40 mV. Notably in no instance was the BK_Ca channel activated by histamine under physiological conditions. The most parsimonious explanation based on the different K_D(Ca) for the two K_Ca channels is provided.

Elevated hydrostatic pressure activates sodium/hydrogen exchanger-1 in rat optic nerve head astrocytes

Mandal, A., Shahidullah, M., Delamere, N. A., Teran, M. A. — 2009-07-01

Optic nerve head astrocytes become abnormal in eyes that have elevated intraocular pressure, and cultured astrocytes display altered protein expression after being subjected for ≥1 days to elevated hydrostatic pressure. Here we show that 2-h elevated hydrostatic pressure (15 or 30 mmHg) causes phosphorylation of ERK1/2, ribosomal S6 protein kinase (p90^RSK), and Na/H exchanger (NHE)1 in cultured rat optic nerve head astrocytes as judged by Western blot analysis. The MEK/ERK inhibitor U0126 abolished phosphorylation of NHE1 and p90^RSK as well as ERK1/2. To examine NHE1 activity, cytoplasmic pH (pH_i) was measured with BCECF and, in some experiments, cells were acidified by 5-min exposure to 20 mM ammonium chloride. Although baseline pH_i was unaltered, the rate of pH_i recovery from acidification was fourfold higher in pressure-treated astrocytes. In the presence of either U0126 or dimethylamiloride (DMA), an NHE inhibitor, hydrostatic pressure did not change the rate of pH_i recovery. The findings are consistent with NHE1 activation due to phosphorylation of ERK1/2, p90^RSK, and NHE1 that occurs in response to hydrostatic pressure. These responses may precede long-term changes of protein expression known to occur in pressure-stressed astrocytes.

Green tea (-)-epigallocatechin gallate inhibits insulin stimulation of 3T3-L1 preadipocyte mitogenesis via the 67-kDa laminin receptor pathway

2009-07-01

Insulin and (–)-epigallocatechin gallate (EGCG) have been reported to regulate fat cell mitogenesis and adipogenesis, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated mitogenesis in 3T3-L1 preadipocytes. EGCG inhibited insulin stimulation of preadipocyte proliferation in a dose- and time-dependent manner. EGCG also suppressed insulin-stimulated phosphorylation of the insulin receptor-β, insulin receptor (IR) substrates 1 and 2 (IRS1 and IRS2), and mitogen-activated protein kinase pathway proteins, RAF1, MEK1/2, and ERK1/2, but not JNK. Furthermore, EGCG inhibited the association of IR with the IRS1 and IRS2 proteins, but not with the IRS4 protein. These data suggest that EGCG selectively affects particular types of IRS and MAPK family members. Generally, EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in modulating insulin-stimulated mitogenic signaling. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and found that its expression was sensitive to growth phase, tissue type, and differentiation state. Pretreatment of preadipocytes with 67LR antiserum prevented the effects of EGCG on insulin-stimulated phosphorylation of IRS2, RAF1, and ERK1/2 and insulin-stimulated preadipocyte proliferation (cell number and bromodeoxyuridine incorporation). Moreover, EGCG tended to increase insulin-stimulated associations between the 67LR and IR, IRS1, IRS2, and IRS4 proteins. These data suggest that EGCG mediates anti-insulin signaling in preadipocyte mitogenesis via the 67LR pathway.

Protein tyrosine phosphatase-{alpha} complexes with the IGF-I receptor and undergoes IGF-I-stimulated tyrosine phosphorylation that mediates cell migration

2009-07-01

Protein tyrosine phosphatase- (PTP) is a widely expressed receptor-type phosphatase that functions in multiple signaling systems. The actions of PTP can be regulated by its phosphorylation on serine and tyrosine residues, although little is known about the conditions that promote PTP phosphorylation. In this study, we tested the ability of several extracellular factors to stimulate PTP tyrosine phosphorylation. The growth factors IGF-I and acidic FGF induced the highest increase in PTP phosphorylation at tyrosine 789, followed by PMA and lysophosphatidic acid, while EGF had little effect. Further investigation of IGF-I-induced PTP tyrosine phosphorylation demonstrated that this occurs through a novel Src family kinase-independent mechanism that does not require focal adhesion kinase, phosphatidylinositol 3-kinase, or MEK. We also show that PTP physically interacts with the IGF-I receptor. In contrast to IGF-I-induced PTP phosphorylation, this association does not require IGF-I. The interaction of PTP and the IGF-I receptor is independent of PTP catalytic activity, and expression of exogenous PTP does not promote IGF-I receptor tyrosine dephosphorylation, indicating that PTP does not act as an IGF-I receptor phosphatase. However, PTP mediates IGF-I signaling, because IGF-I-stimulated fibroblast migration was reduced by ~50% in cells lacking PTP or in cells with mutant PTP lacking the tyrosine 789 phosphorylation site. Our results suggest that PTP tyrosine phosphorylation can occur in response to diverse stimuli and can be mediated by various tyrosine kinases. In the case of IGF-I, we propose that IGF-I-induced tyrosine 789 phosphorylation of PTP, possibly catalyzed by the PTP-associated IGF-I receptor tyrosine kinase, is required for efficient cell migration in response to this growth factor.

Downregulation of cilia-localized Il-6R{alpha} by 17{beta}-estradiol in mouse and human fallopian tubes

2009-07-01

The action of interleukin-6 (IL-6) impacts female reproduction. Although IL-6 was recently shown to inhibit cilia activity in human fallopian tubes in vitro, the molecular mechanisms underlying IL-6 signaling to tubal function remain elusive. Here, we investigate the cellular localization, regulation, and possible function of two IL-6 receptors (IL-6R and gp130) in mouse and human fallopian tubes in vivo. We show that IL-6R is restricted to the cilia of epithelial cells in both mouse and human fallopian tubes. Exogenous 17β-estradiol (E₂), but not progesterone (P₄), causes a time-dependent decrease in IL-6R expression, which is blocked by the estrogen receptor (ER) antagonist ICI-182,780. Exposure of different ER-selective agonists propyl-(1H)-pyrazole-1,3,5-triyl-trisphenol or 2,3-bis-(4-hydroxyphenyl)-propionitrile demonstrated an ER subtype-specific regulation of IL-6R in mouse fallopian tubes. In contrast to IL-6R, gp130 was detected in tubal epithelial cells in mice but not in humans. In humans, gp130 was found in the muscle cells and was decreased in the periovulatory and luteal phases during the reproductive cycles, indicating a species-specific expression and regulation of gp130 in the fallopian tube. Expression of tubal IL-6R and gp130 in IL-6 knockout mice was found to be normal; however, E₂ treatment increased IL-6R, but not gp130, in IL-6 knockout mice when compared with wild-type mice. Furthermore, expression levels of IL-6R, but not gp130, decreased in parallel with estrogenic accelerated oocyte-cumulus complex (OCC) transport in mouse fallopian tubes. Our findings open the posibility that cilia-specific IL-6R may play a role in the regulation of OCC transport and suggest an estrogen-regulatory pathway of IL-6R in the fallopian tube.

Facilitated maturation of Ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression

Liu, J., Lieu, D. K., Siu, C. W., Fu, J.-D., Tse, H.-F., Li, R. A. — 2009-07-01

Cardiomyocytes (CMs) are nonregenerative. Self-renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell-based therapies. We recently reported that Ca²⁺ handling, crucial to excitation-contraction coupling of hESC-derived CMs (hESC-CMs), is functional but immature. Such immature properties as smaller cytosolic Ca²⁺ transient amplitudes, slower kinetics, and reduced Ca²⁺ content of sarcoplasmic reticulum (SR) can be attributed to the differential developmental expression profiles of specific Ca²⁺ handling and regulatory proteins in hESC-CMs and their adult counterparts. In particular, calsequestrin (CSQ), the most abundant, high-capacity but low-affinity, Ca²⁺-binding protein in the SR that is anchored to the ryanodine receptor, is robustly expressed in adult CMs but completely absent in hESC-CMs. Here we hypothesized that gene transfer of CSQ in hESC-CMs suffices to induce functional improvement of SR. Transduction of hESC-CMs by the recombinant adenovirus Ad-CMV-CSQ-IRES-GFP (Ad-CSQ) significantly increased the transient amplitude, upstroke velocity, and transient decay compared with the control Ad-CMV-GFP (Ad-GFP) and Ad-CMV-CSQ-IRES-GFP (Ad-CSQ, which mediated the expression of a nonfunctional, truncated version of CSQ) groups. Ad-CSQ increased the SR Ca²⁺ content but did not alter L-type Ca²⁺ current. Pharmacologically, untransduced wild-type, Ad-GFP-, Ad-CSQ-, and Ad-CSQ-transduced hESC-CMs behaved similarly. Whereas ryanodine significantly reduced the Ca²⁺ transient amplitude and slowed the upstroke, thapsigargin slowed the decay. Neither triadin nor junctin was affected. We conclude that CSQ expression in hESC-CMs facilitates Ca²⁺ handling maturation. Our results shed insights into the suitability of hESC-CMs for therapies and as certain heart disease models for drug screening.

The dyslexia-associated protein KIAA0319 interacts with adaptor protein 2 and follows the classical clathrin-mediated endocytosis pathway

Levecque, C., Velayos-Baeza, A., Holloway, Z. G., Monaco, A. P. — 2009-07-01

Recently, genetic studies have implicated KIAA0319 in developmental dyslexia, the most common of the childhood learning disorders. The first functional data indicated that the KIAA0319 protein is expressed on the plasma membrane and may be involved in neuronal migration. Further analysis of the subcellular distribution of the overexpressed protein in mammalian cells indicates that KIAA0319 can colocalize with the early endosomal marker early endosome antigen 1 (EEA1) in large intracellular vesicles, suggesting that it is endocytosed. Antibody internalization assays with full-length KIAA0319 and deletion constructs confirmed that KIAA0319 is internalized and showed the importance of the cytoplasmic juxtamembranal region in this process. The present study has identified the medium subunit (µ2) of adaptor protein 2 (AP-2) as a binding partner of KIAA0319 in a yeast two-hybrid screen. Using Rab5 mutants or depletion of the µ-subunit of AP-2 or clathrin heavy chain by RNA interference, we demonstrate that KIAA0319 follows a clathrin-mediated endocytic pathway. We also identify tyrosine-995 of KIAA0319 as a critical amino acid required for the interaction with AP-2 and subsequent internalization. These results suggest the surface expression of KIAA0319 is regulated by endocytosis, supporting the idea that the internalization and recycling of the protein may be involved in fine tuning its role in neuronal migration.

Transfer of ascorbic acid across the vascular endothelium: mechanism and self-regulation

May, J. M., Qu, Z.-c., Qiao, H. — 2009-07-01

To determine how ascorbic acid moves from the bloodstream into tissues, we assessed transfer of the vitamin across the barrier generated by EA.hy926 endothelial cells when these were cultured on semipermeable filter supports. Ascorbate transfer from the luminal to the abluminal compartment was time dependent, inhibited by anion channel blockers and by activation of protein kinase A, but was increased by thrombin. Ascorbate transfer occurred by a paracellular route, since it did not correlate with intracellular ascorbate contents and was not rectified or saturable. Nonetheless, intracellular ascorbate inhibited the transfer of both ascorbate and radiolabeled inulin across the endothelial barrier. The increase in barrier function due to ascorbate was dependent on its intracellular concentration, significant by 15 min of incubation, prevented by the cytoskeletal inhibitor colchicine, associated with F-actin stress fiber formation, and not due to collagen deposition. These results show that ascorbate traverses the endothelial barrier by a paracellular route that is regulated by cell metabolism, ion channels, and ascorbate itself. Since the latter effect occurred over the physiological range of ascorbate plasma concentrations, it could reflect a role for the vitamin in control of endothelial barrier function in vivo.

Endothelial cell traction and ECM density influence both capillary morphogenesis and maintenance in 3-D

Kniazeva, E., Putnam, A. J. — 2009-07-01

Identifying the mechanisms regulating angiogenesis in pathological conditions such as cancer and heart disease is crucial to develop successful therapies. The dependence of angiogenesis on characteristic properties of these conditions, such as alterations in tissue stiffness due to changes in the composition of the extracellular matrix (ECM), may shed light on potential therapeutic strategies. Prior studies have suggested that ECM compliance regulates capillary morphogenesis, but the mechanisms remain unclear. In this study, we hypothesized that ECM density, which influences substrate mechanics, may regulate angiogenesis via a mechanism involving actin-mediated cell-generated forces. To investigate this hypothesis, we utilized an in vitro model of angiogenesis in which endothelial cells coated on microcarrier beads are distributed within a three-dimensional (3-D) fibrin ECM. A monolayer of fibroblasts, which provides pro-angiogenic factors, is cultured on top of the gel. Variations in fibrin gel density, along with a library of pharmacological agents that inhibit forces generated by the actin cytoskeleton, were used to prove the necessity of cell-generated tractional forces in blood vessel formation. Our data demonstrate that cell-generated forces not only play a crucial role in the early sprouting stages of capillary morphogenesis but are also required in the later maintenance stages, and thereby suggest a broader interdependence among tissue stiffness, cell contractile forces, and angiogenesis.

Identification of the large-conductance background K+ channel in mouse B cells as TREK-2

2009-07-01

Mouse B cells and their cell line (WEHI-231) express large-conductance background K⁺ channels (LK_bg) that are activated by arachidonic acids, characteristics similar to TREK-2. However, there is no evidence to identify the molecular nature of LK_bg; some properties of LK_bg were partly different from the reported results of TREK type channels. In this study, we compared the properties of cloned TREK-2 and LK_bg in terms of their sensitivities to ATP, phosphatidylinositol 4,5-bisphosphate (PIP₂), intracellular pH (pH_i), and membrane stretch. Similar to the previous findings of LK_bg, TREK-2 showed spontaneous activation after membrane excision (i-o patch) and were inhibited by MgATP or by PIP₂. The inhibition by MgATP was prevented by wortmannin, suggesting membrane-delimited regulation of TREKs by phosphoinositide (PI) kinase. The same was observed with the property of LK_bg; the activation of TREK-2 by membrane stretch was suppressed by U73122 (PLC inhibitor). As with the known properties of TREK-2, LK_bg were activated by acidic pH_i and inhibited by PKC activator. Finally, we confirmed the expression of TREK-2 in WEHI-231 by using RT-PCR and immunoblot analyses. The amplitude of background K⁺ current and the TREK-2 expression in WEHI-231 were commonly decreased by genetic knockdown of TREK-2 using small interfering RNA. The downregulation of TREK-2 attenuated Ca²⁺-influx induced by arachidonic acid in WEHI-231. As a whole, these results strongly indicate that TREK-2 encodes LK_bg in mouse B cells. We also newly suggest that the low activity of TREK-2 in intact cells is due to the inhibition by intrinsic PIP₂.

ROS activate KCl cotransport in nonadherent Ehrlich ascites cells but K+ and Cl- channels in adherent Ehrlich Lettre and NIH3T3 cells

Lambert, I. H., Klausen, T. K., Bergdahl, A., Hougaard, C., Hoffmann, E. K. — 2009-07-01

Addition of H₂O₂ (0.5 mM) to Ehrlich ascites tumor cells under isotonic conditions results in a substantial (22 ± 1%) reduction in cell volume within 25 min. The cell shrinkage is paralleled by net loss of K⁺, which was significant within 8 min, whereas no concomitant increase in the K⁺ or Cl^– conductances could be observed. The H₂O₂-induced cell shrinkage was unaffected by the presence of clofilium and clotrimazole, which blocks volume-sensitive and Ca²⁺-activated K⁺ channels, respectively, and is unaffected by a raise in extracellular K⁺ concentration to a value that eliminates the electrochemical driving force for K⁺. On the other hand, the H₂O₂-induced cell shrinkage was impaired in the presence of the KCl cotransport inhibitor (dihydro-indenyl)oxyalkanoic acid (DIOA), following substitution of NO₃^– for Cl^–, and when the driving force for KCl cotransport was omitted. It is suggested that H₂O₂ activates electroneutral KCl cotransport in Ehrlich ascites tumor cells and not K⁺ and Cl^– channels. Addition of H₂O₂ to hypotonically exposed cells accelerates the regulatory volume decrease and the concomitant net loss of K⁺, whereas no additional increase in the K⁺ and Cl^– conductance was observed. The effect of H₂O₂ on cell volume was blocked by the serine-threonine phosphatase inhibitor calyculin A, indicating an important role of serine-threonine phosphorylation in the H₂O₂-mediated activation of KCl cotransport in Ehrlich cells. In contrast, addition of H₂O₂ to adherent cells, e.g., Ehrlich Lettré ascites cells, a subtype of the Ehrlich ascites tumor cells, and NIH3T3 mouse fibroblasts increased the K⁺ and Cl^– conductances after hypotonic cell swelling. Hence, H₂O₂ induces KCl cotransport or K⁺ and Cl^– channels in nonadherent and adherent cells, respectively.

Arachidonic acid potentiates hypoxia-induced VEGF expression in mouse embryonic stem cells: involvement of Notch, Wnt, and HIF-1{alpha}

Lee, S. H., Kim, M. H., Han, H. J. — 2009-07-01

Recent investigations suggest that hypoxia increases the release of fatty acids, which participate in the regulation of cytokine synthesis and cell growth. Therefore, in this study, we examined the effect of arachidonic acid (AA) on hypoxia-induced vascular endothelial growth factor (VEGF) expression and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia increased the level of [³H]AA release and VEGF expression. AA treatment concurrent with hypoxia further increased the PGE₂ production and VEGF expression level, which was inhibited by the suppression of cPLA₂ and cyclooxygenase 2 (COX-2) pathways. Hypoxia increased the level of Notch-1 and Wnt-1/β-catenin expression, which was blocked by the inhibition of COX-2, and inhibition of Notch-1 by -secretase inhibitor blocked Wnt-1 activation. Moreover, the hypoxia-induced increase of hypoxia-inducible factor 1 (HIF-1) expression induced Notch-1 activation and was regulated by Wnt-1 activation. The expression of each signaling molecule induced an increase in VEGF expression that was greater in hypoxia with AA than in hypoxia alone. The inhibition of VEGF expression using VEGF-targeted small interfering RNA decreased the hypoxia-induced increase in cell cycle regulatory protein expression, DNA synthesis, and cell number, suggesting that hypoxia-induced VEGF expression stimulates proliferation of mouse ES cells. In conclusion, AA potentiates hypoxia-induced VEGF expression in mouse ES cells through the Notch-1, Wnt-1, and HIF-1 pathways.

The role of PGC-1{alpha} on mitochondrial function and apoptotic susceptibility in muscle

2009-07-01

Mitochondria are critical for cellular bioenergetics, and they mediate apoptosis within cells. We used whole body peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) knockout (KO) animals to investigate its role on organelle function, apoptotic signaling, and cytochrome-c oxidase activity, an indicator of mitochondrial content, in muscle and other tissues (brain, liver, and pancreas). Lack of PGC-1 reduced mitochondrial content in all muscles (17–44%; P < 0.05) but had no effect in brain, liver, and pancreas. However, the tissue expression of proteins involved in mitochondrial DNA maintenance [transcription factor A (Tfam)], import (Tim23), and remodeling [mitofusin 2 (Mfn2) and dynamin-related protein 1 (Drp1)] did not parallel the decrease in mitochondrial content in PGC-1 KO animals. These proteins remained unchanged or were upregulated (P < 0.05) in the highly oxidative heart, indicating a change in mitochondrial composition. A change in muscle organelle composition was also evident from the alterations in subsarcolemmal and intermyofibrillar mitochondrial respiration, which was impaired in the absence of PGC-1. However, endurance-trained KO animals did not exhibit reduced mitochondrial respiration. Mitochondrial reactive oxygen species (ROS) production was not affected by the lack of PGC-1, but subsarcolemmal mitochondria from PGC-1 KO animals released a greater amount of cytochrome c than in WT animals following exogenous ROS treatment. Our results indicate that the lack of PGC-1 results in 1) a muscle type-specific suppression of mitochondrial content that depends on basal oxidative capacity, 2) an alteration in mitochondrial composition, 3) impaired mitochondrial respiratory function that can be improved by training, and 4) a greater basal protein release from subsarcolemmal mitochondria, indicating an enhanced mitochondrial apoptotic susceptibility.

Corrigendum

2009-07-01