Alla F. Fomina

Massive Secretion by T Cells Is Caused by HIV Nef in Infected Cells and by Nef Transfer to Bystander Cells

The HIV Nef protein mediates endocytosis of surface receptors that correlates with disease progression, but the link between this Nef function and HIV pathogenesis is not clear. Here, we report that Nef-mediated activation of membrane trafficking is bidirectional, connecting endocytosis with exocytosis as occurs in activated T cells. Nef expression induced an extensive secretory activity in infected and, surprisingly, also in noninfected T cells, leading to the massive release of microvesicle clusters, a phenotype observed in vitro and in 36%-87% of primary CD4 T cells from HIV-infected individuals. Consistent with exocytosis in noninfected cells, Nef is transferred to bystander cells upon cell-to-cell contact and subsequently induces secretion in an Erk1/2-dependent manner. Thus, HIV Nef alters membrane dynamics, mimicking those of activated T cells and causing a transfer of infected cell signaling (TOS) to bystander cells. This mechanism may help explain the detrimental effect on bystander cells seen in HIV infection.

Dexamethasone rapidly induces Kv1.5 K+ channel gene transcription and expression in clonal pituitary cells

Glucocorticoids specifically increase Kv1.5 K+ channel mRNA in normal and clonal (GH3) rat pituitary cells. Here, we demonstrate that dexamethasone, a glucocorticoid agonist, rapidly induces Kv1.5 gene transcription, but does not affect Kv1.5 mRNA turnover (t1/2 approximately 0.5 hr) in GH3 cells. Immunoblots indicate that the steroid also increases the expression of the 76 kd Kv1.5 protein approximately 3-fold within 12 hr without altering its half-life (t1/2 approximately 4 hr). In contrast, Kv1.4 protein expression is unaffected. Finally, we find that the induction of Kv1.5 protein is associated with an increase in a noninactivating component of the voltage-gated K+ current. Our results indicate that hormones and neurotransmitters may act within hours to regulate excitability by controlling K+ channel gene expression.

T cell exosomes induce cholesterol accumulation in human monocytes via phosphatidylserine receptor.

Activated T lymphocytes release vesicles, termed exosomes, enriched in cholesterol and exposing phosphatidylserine (PS) at their outer membrane leaflet. Although CD4+ activated T lymphocytes infiltrate an atherosclerotic plaque, the effects of T cell exosomes on the atheroma‐associated cells are not known. We report here that exosomes isolated from the supernatants of activated human CD4+ T cells enhance cholesterol accumulation in cultured human monocytes and THP‐1 cells. Lipid droplets found in the cytosol of exosome‐treated monocytes contained both cholesterol ester and free cholesterol. Anti‐phosphatidylserine receptor antibodies recognized surface protein on the monocyte plasma membrane and prevented exosome‐induced cholesterol accumulation, indicating that exosome internalization is mediated via endogenous phosphatidylserine receptor. The production of proinflammatory cytokine TNF‐α enhanced in parallel with monocyte cholesterol accumulation. Our data strongly indicate that exosomes released by activated T cells may represent a powerful, previously unknown, atherogenic factor. J. Cell. Physiol. 212: 174–181, 2007.

Endogenous expression of TRPV5 and TRPV6 calcium channels in human leukemia K562 cells

In blood cells, changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) are associated with multiple cellular events, including activation of cellular kinases and phosphatases, degranulation, regulation of cytoskeleton binding proteins, transcriptional control, and modulation of surface receptors. Although there is no doubt as to the significance of Ca(2+) signaling in blood cells, there is sparse knowledge about the molecular identities of the plasmalemmal Ca(2+) permeable channels that control Ca(2+) fluxes across the plasma membrane and mediate changes in [Ca(2+)](i) in blood cells. Using RNA expression analysis, we have shown that human leukemia K562 cells endogenously coexpress transient receptor potential vanilloid channels type 5 (TRPV5) and type 6 (TRPV6) mRNAs. Moreover, we demonstrated that TRPV5 and TRPV6 channel proteins are present in both the total lysates and the crude membrane preparations from leukemia cells. Immunoprecipitation revealed that a physical interaction between TRPV5 and TRPV6 may take place. Single-channel patch-clamp experiments demonstrated the presence of inwardly rectifying monovalent currents that displayed kinetic characteristics of unitary TRPV5 and/or TRPV6 currents and were blocked by extracellular Ca(2+) and ruthenium red. Taken together, our data strongly indicate that human myeloid leukemia cells coexpress functional TRPV5 and TRPV6 calcium channels that may interact with each other and contribute into intracellular Ca(2+) signaling.

Store-operated Ca2+ Influx Causes Ca2+ Release from the Intracellular Ca2+ Channels That Is Required for T Cell Activation

The precise control of many T cell functions relies on cytosolic Ca2+ dynamics that is shaped by the Ca2+ release from the intracellular store and extracellular Ca2+ influx. The Ca2+ influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca2+ concentration ([Ca2+]i) necessary for T cell activation, whereas the role of intracellular Ca2+ release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca2+]i elevation observed upon activation of the store-operated Ca2+ entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca2+-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca2+ release channels were activated in parallel with SOCE and contributed to global [Ca2+]i elevation. Pretreating cells with (-)-xestospongin C (10 μm) or ryanodine (400 μm), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca2+]i elevation evoked by the passive store depletion or TCR ligation. Although the Ca2+ release from the IP3R can be activated by TCR stimulation, the Ca2+ release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca2+]i signaling in T cells, that is SOCE-dependent Ca2+ release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca2+-dependent functions of T lymphocytes.

Dexamethasone rapidly increases calcium channel subunit messenger rna expression and high voltage-activated calcium current in clonal pituitary cells

Glucocorticoid hormones increase voltage-gated Ca(2)+ current density in clonal pituitary cells. To test whether these steroids might stimulate expression of Ca(2)+ channel genes, messenger RNase protection assays were used to measure alpha IC and alpha ID RNAs that encode pore-forming subunits of L-type Ca2+ channels. We show here that dexamethasone rapidly increases alpha IC messenger RNA expression without affecting alpha ID messenger RNA level. This up-regulation of channel messenger RNA is also produced by natural glucocorticoids and is blocked by the glucocorticoid antagonist Ru48386. The up-regulation of the channel subunit messenger RNA expression is associated with an increase in high voltage-activated Ca(2)+ current density. Thus, glucocorticoids may produce a long-term effect on Ca(2)+ homeostasis in clonal pituitary cells by differentially regulating expression of Ca(2)+ channel subunit genes.

Control of Ca2+ channel current and exocytosis in rat lactotrophs by basally active protein kinase C and calcineurin

Modulation of voltage-activated Ca2+ channel activity by phosphorylation was studied in metabolically intact voltage-clamped rat lactotrophs. Experiments using Ba2+ as a charge carrier indicated that a phorbol ester protein kinase C activator stimulates high-voltage-activated Ca2+ channel currents, but has no effect on low-voltage-activated currents. Extracellular application of structurally and mechanistically distinct protein kinase C inhibitors (staurosporin, H7, calphostin C, chelerythrine and Ro 31-8220) preferentially inhibited the high-voltage-activated Ba2+ current. This suggests that protein kinase C is required for maintainance of Ca2+ channel activity even in the absence of modulators. Cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, increased the high-voltage-activated Ca2+ channel current, and staurosporin reversed this effect. Thus, dephosphosphorylation by calcineurin may limit basal Ca2+ channel activity. Time-domain monitoring of cellular capacitance changes demonstrated that cyclosporin A and 12-O-tetradecanoyl-phorbol-13-acetate do not affect exocytosis at a hyperpolarized potential, but each enhances depolarization-induced exocytosis. Facilitation of exocytosis by cyclosporin A differed from 12-O-tetradecanoyl-phorbol-13-acetate in that it was biphasic. The delayed facilitation induced by cyclosporin A could be accounted for by stimulation of the voltage-gated Ca2+ current. These results suggest that the high-voltage activated Ca2+ channel current in rat lactotrophs is determined by the opposing basal activities of protein kinase C and calcineurin. Furthermore, it is concluded that the regulation of Ca2+ channels by protein kinase C and calcineurin affects depolarization-induced exocytosis.

Bidirectional Coupling between Ryanodine Receptors and Ca2+ Release-activated Ca2+ (CRAC) Channel Machinery Sustains Store-operated Ca2+ Entry in Human T Lymphocytes

Background: Ryanodine receptors (RyR) are intracellular Ca2+ release channels. Results: RyR are expressed in human T cells. Modulating the RyR activity in human T cells affects Ca2+ leakage from the store and store-operated Ca2+ entry. Conclusion: RyR strongly regulate Ca2+ influx in human T cells. Significance: RyR can be used as a target for manipulating immune responses in humans. The expression and functional significance of ryanodine receptors (RyR) were investigated in resting and activated primary human T cells. RyR1, RyR2, and RyR3 transcripts were detected in human T cells. RyR1/2 transcript levels increased, whereas those of RyR3 decreased after T cell activation. RyR1/2 protein immunoreactivity was detected in activated but not in resting T cells. The RyR agonist caffeine evoked Ca2+ release from the intracellular store in activated T cells but not in resting T cells, indicating that RyR are functionally up-regulated in activated T cells compared with resting T cells. In the presence of store-operated Ca2+ entry (SOCE) via plasmalemmal Ca2+ release-activated Ca2+ (CRAC) channels, RyR blockers reduced the Ca2+ leak from the endoplasmic reticulum (ER) and the magnitude of SOCE, suggesting that a positive feedback relationship exists between RyR and CRAC channels. Overexpression of fluorescently tagged RyR2 and stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor gating CRAC channels, in HEK293 cells revealed that RyR are co-localized with STIM1 in the puncta formed after store depletion. These data indicate that in primary human T cells, the RyR are coupled to CRAC channel machinery such that SOCE activates RyR via a Ca2+-induced Ca2+ release mechanism, which in turn reduces the Ca2+ concentration within the ER lumen in the vicinity of STIM1, thus facilitating SOCE by reducing store-dependent CRAC channel inactivation. Treatment with RyR blockers suppressed activated T cell expansion, demonstrating the functional importance of RyR in T cells.

Intracellular Ca2+ Release Triggers Translocation of Membrane Marker FM1–43 from the Extracellular Leaflet of Plasma Membrane into Endoplasmic Reticulum in T Lymphocytes

Stimulation of T cell receptor in lymphocytes enhances Ca2+ signaling and accelerates membrane trafficking. The relationships between these processes are not well understood. We employed membrane-impermeable lipid marker FM1–43 to explore membrane trafficking upon mobilization of intracellular Ca2+ in Jurkat T cells. We established that liberation of intracellular Ca2+ with T cell receptor agonist phytohemagglutinin P or with Ca2+-mobilizing agents ionomycin or thapsigargin induced accumulation of FM1–43 within the lumen of the endoplasmic reticulum (ER), nuclear envelope (NE), and Golgi. FM1–43 loading into ER-NE and Golgi was not mediated via the cytosol because other organelles such as mitochondria and multivesicular bodies located in close proximity to the FM1–43-containing ER were free of dye. Intralumenal FM1–43 accumulation was observed even when Ca2+ signaling in the cytosol was abolished by the removal of extracellular Ca2+. Our findings strongly suggest that release of intracellular Ca2+ may create continuity between the extracellular leaflet of the plasma membrane and the lumenal membrane leaflet of the ER by a mechanism that does not require global cytosolic Ca2+ elevation.

Endogenous Jmjd6 gene product is expressed at the cell surface and regulates phagocytosis in immature monocyte-like activated THP-1 cells.

A jumonji domain containing gene 6 (Jmjd6), previously referred to as phosphatidylserine receptor (PSR) gene, plays an important role in cell differentiation and development of multiple organs, although mechanisms of its action are not known. The Jmjd6 gene product was initially identified as a membrane protein that participates in phagocytosis. However, the later findings that recombinant Jmjd6 in expression systems was targeted to the nucleus challenged the role of Jmjd6 as a membrane receptor. Using immunocytochemistry approach we studied the subcellular distribution of endogenous Jmjd6 protein in THP‐1 cells activated with phorbol 12‐myristate 13 acetate (PMA). We found that treatment with PMA stimulated Jmjd6 expression in the cytosol of activated cells. Furthermore, Jmjd6 initially appeared at the cell surface of immature phagocytes (1–2 days after activation) but then translocated into the nucleus of differentiated macrophage‐like cells (5–9 days after activation). Anti‐Jmjd6 antibodies suppressed the engulfment of dead cell corpses by THP‐1 cells expressing the Jmjd6 at the cell surface. These data indicate that Jmjd6 serves as a membrane‐associated receptor that regulates phagocytosis in immature macrophages but is dispensable for phagocytosis and has other functions when it is expressed in the cytosol and nucleus of mature macrophage‐like cells. J. Cell. Physiol. 221: 84–91, 2009.