I. I. Marakhova

Na, K-ATPase pump in activated human lymphocytes: on the mechanisms of rapid and long-term increase in K influxes during the initiation of phytohemagglutinin-induced proliferation.

Functional expression of Na, K-ATPase pump as determined by ouabain-sensitive Rb influxes has been investigated in human peripheral blood lymphocytes, activated by phytohemagglutinin (PHA) from resting state to proliferation. It is found that a rapid twofold elevation of ouabain-sensitive Rb influx in response to PHA is followed by a long-term increase in pump activity, which precedes the DNA synthesis and is temporally related to the growth phase of mitogenic response. Unlike the early pump activation, the late enhanced pump activity is not the result of elevated cell Na content, it is inhibited by cycloheximide and requires new protein synthesis. Actinomycin D and alpha-amanitin, in doses, which suppress the PHA-induced increase in the RNA synthesis, do not abolish the elevated Rb influx until 20-24h of mitogenic activation and inhibit the late, growth-associated increase in Rb influx. It is concluded that (1) in mitogen-activated cells both short- and long-term control is involved in the enhanced pump activity, and (2) translational and transcriptional mechanisms may contribute to the long-term up-regulation of Na, K-ATPase pump during blast transformation of human lymphocytes.

Expression of transient receptor potential vanilloid channels TRPV5 and TRPV6 in human blood lymphocytes and Jurkat leukemia T cells.

Regulation of Ca2+ entry is a key process for lymphocyte activation, cytokine synthesis and proliferation. Several members of the transient receptor potential (TRP) channel family can contribute to changes in [Ca2+]in; however, the properties and expression levels of these channels in human lymphocytes continue to be elusive. Here, we established and compared the expression of the most Ca2+-selective members of the TRPs, Ca2+ channels transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6), in human blood lymphocytes (HBLs) and leukemia Jurkat T cells. We found that TRPV6 and TRPV5 mRNAs are expressed in both Jurkat cells and quiescent HBLs; however, the levels of mRNAs were significantly higher in malignant cells than in quiescent lymphocytes. Western blot analysis showed TRPV5/V6 proteins in Jurkat T cells and TRPV5 protein in quiescent HBLs. However, the expression of TRPV6 protein was switched off in quiescent HBLs and turned on after mitogen stimulation of the cells with phytohemagglutinin. Inwardly directed monovalent currents that displayed characteristics of TRPV5/V6 currents were recorded in both Jurkat cells and normal HBLs. In outside–out patch-clamp studies, currents were reduced by ruthenium red, a nonspecific inhibitor of TRPV5/V6 channels. In addition, ruthenium red downregulated cell-cycle progression in both activated HBLs and Jurkat cells. Thus, we identified TRPV5 and TRPV6 calcium channels, which can be considered new candidates for Ca2+ entry into human lymphocytes. The correlation between expression of TRPV6 channels and the proliferative status of lymphocytes suggests that TRPV6 may be involved in the physiological and/or pathological proliferation of lymphocytes.

Interleukin‐2‐dependent regulation of Na/K pump in human lymphocytes

The present study provides the first evidence that the abundance of catalytic α1‐subunit of Na,K‐ATPase increases in the course of T cell blast transformation. Immunodepressant cyclosporin A at anti‐proliferative doses diminished the induction of α1 protein in activated lymphocytes. Furthermore, in competent T cells, IL‐2 increases both the transport activity of Na/K pump and the content of Na,K‐ATPase α1 protein in a time‐dependent manner. A correlation was found between the long‐term elevation in ouabain‐sensitive Rb influxes and the increase in α1 protein content in late activated T cells. These results suggest that (1) the increased expression of Na,K‐ATPase proteins underlie the cell cycle‐dependent upregulation of ion pump during T cell transformation, and (2) IL‐2 is involved in the regulated expression of Na,K‐ATPase in human lymphocytes.

Expression of mRNAs encoding the α1 and the β1 subunits of Na+,K+-ATPase in human lymphocytes activated with phytohaemagglutinine

Increase in Na+,K+‐ATPase mRNAs was detected in activated lymphocytes by the RT‐PCR method. α1 subunit mRNA gradualy increased with time and by 36 h was 2.4 times higher than at the start. Increase in the β1 mRNA was transient reaching a maximum in the 8 h probe and declining to the initial level in the 24 and 36 h probes. The elevation of Na+,K−‐ATPase mRNAs does not underlie a cycloheximide‐inhibited increase in cation pumping peculiar to the prereplicative period as can be judged from the fact that Act D fails to eliminate PHA‐induced enhancement of pump fluxes.

Long-term regulation of Na,K-ATPase pump during T-cell proliferation

The aim of the study was to elucidate the mechanism responsible for the proliferation-related regulation of Na,K-ATPase pump. Our data demonstrate that in mitogen-stimulated human blood lymphocytes, enhanced ouabain-sensitive Rb(K) fluxes in the middle/late stage of G0/G1/S transit are associated with the increased number of Na,K-ATPase pumps expressed at the cell surface (as determined by the [3H]ouabain binding). Analysis of total RNA (reverse transcription-polymerase chain reaction) and protein (Western blotting) showed a threefold increase in the level of Na,K-ATPase α1-subunit and β1-subunit mRNAs and significant increase in the Na,K-ATPase α1-subunit protein during the first day of mitogen-induced proliferation. The elevated K transport as well as the increased expression of Na,K-ATPase is closely associated with the IL-2-dependent stage of T-cell response. The pharmacological inhibition of IL-2-induced MEK/ERK or JAK/STAT cascades suppressed the IL-2-induced proliferation and reduced the functional and protein expressions of Na,K-ATPase. It is concluded that during the lymphocyte transition from resting stage to proliferation, (1) long-term activation of Na,K-ATPase pump is due to the enhanced expression of Na,K-ATPase protein and mRNA, and (2) the cytokine signaling via the IL-2 receptor is necessary for the cell cycle-associated upregulation of Na,K-ATPase.

TRPV5 and TRPV6 calcium channels in human T cells

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) has provided a molecular basis for studying previously unidentified calcium influx channels in electrically nonexcitable cells. In the present work using RT-PCR, we obtained the endogenous expression of mRNAs of genes trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in normal human T lymphocytes. Additionally, by immunoblotting, the presence of the channel-forming TRPV5 proteins has been shown both in the total lysate and in crude membrane fractions from Jurkat cells and normal T lymphocytes. The use of immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes, this protein was not detected. The expression pattern and the selective Ca2+ permeation properties of TRPV5 and TRPV6 channels indicate the important role of these channels in Ca2+ homeostasis, as well as most likely in malignant transformation of blood cells.

Long-term enhancement of Na,K-ATPase pump during blasttransformation of human lymphocytes is controlled first by translational, then by transcriptional mechanisms

The transition of phytohemagglutinin‐activated human lymphocytes from resting state to proliferation is accompanied by a long‐term increase in ouabain‐sensitive Rb(K) influx which is closely related to a cyclosporin A‐sensitive step of G0/G1/S progression. At least two distinct phases of the up‐regulation of cation pump has been revealed: the initial stage (5–20 h) which is cycloheximide‐inhibitable and actinomycin D (α‐amanitin)‐unaffected, and the later stage (after 20 h) which is cycloheximide‐ and actinomycin D (α‐amanitin)‐inhibitable. Thus, the enhanced Na,K‐ATPase pump during the cell progression from quiescence to proliferation is controlled both at translational and transcriptional levels.

Functional expression of the Na/K pump is controlled via a cyclosporin A-sensitive signalling pathway in activated human lymphocytes.

An immunosuppressant cyclosporin A (CsA) inhibits T‐cell proliferation by blocking the nuclear factor of activated T‐cells (NFAT) required for expression of the interleukin‐2 (IL‐2) gene. This work has demonstrated for the first time that in human blood lymphocytes (HBLs) activated by phytohemagglutinin (PHA), CsA at anti‐proliferative doses inhibits the late sustained increase in ouabain‐sensitive Rb(K) influxes, which accompanies the growth phase of G0/G1/S transition. CsA affects neither the initial, transient activation of the pump in response to PHA nor the ouabain‐resistant ion fluxes during cell cycle progression. When the HBLs were rendered competent to proliferate by phorbol 12,13‐dibutyrate ester and ionomycin in the presence of CsA, the exogenous IL‐2 did not bypass the initial inhibitory effect of CsA on the long‐term pump enhancement. When applied after the competence induction, CsA produced no effect on the sustained increase in ouabain‐sensitive Rb influxes during the IL‐2‐induced progression phase. These results indicate that in activated HBLs, (1) IL‐2 is involved in functional expression of the Na/K pump during cell transition from quiescence to proliferation, (2) the cell cycle‐associated upregulation of the pump is related to a CsA‐sensitive signalling pathway.

Time-Dependent Regulation of IL-2R α-Chain (CD25) Expression by TCR Signal Strength and IL-2-Induced STAT5 Signaling in Activated Human Blood T Lymphocytes

The expression of the IL-2R α-chain (IL-2Rα) is regulated at the transcriptional level via TCR- and IL-2R-signaling. The question is how to precede in time the activation signals to induce the IL-2Rα expression in native primary T cells. By comparing the effects of selective drugs on the dynamics of CD25 expression during the mitogen stimulation of human peripheral blood lymphocytes, we identified distinct Src- and JAK-dependent stages of IL-2Rα upregulation. PP2, a selective inhibitor of TCR-associated Src kinase, prevents CD25 expression at initial stages of T cell activation, prior to the cell growth. This early IL-2Rα upregulation underlies the T cell competence and the IL-2 responsiveness. We found that the activated with “weak” mitogen, the population of blood lymphocytes has some pool of competent CD25+ cells bearing a high affinity IL-2R. A distinct pattern of IL-2R signaling in resting and competent T lymphocytes has been shown. Based on the inhibitory effect of WHI-P131, a selective drug of JAK3 kinase activity, we concluded that in quiescent primary T lymphocytes, the constitutive STAT3 and the IL-2-induced prolonged STAT5 activity (assayed by tyrosine phosphorylation) is mostly JAK3-independent. In competent T cells, in the presence of IL-2 JAK3/STAT5 pathway is switched to maintain the higher and sustained IL-2Rα expression as well as cell growth and proliferation. We believe that understanding the temporal coordination of antigen- and cytokine-evoked signals in primary T cells may be useful for improving immunotherapeutic strategies.

FUNCTIONAL CHARACTERISTICS OF TRPV5 AND TRPV6 CHANNELS IN NORMAL AND TRANSFORMED HUMAN LYMPHOCYTES

Calcium signaling and Ca2+-conducting channels participate in development of immune response, cell proliferation, growth, and differentiation of lymphocytes. In this paper, the calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) were studied in the plasma membrane of the T cell line Jurkat and normal human blood lymphocytes (HBLs). The channels were spontaneously activated after removal of Ca2+ and Mg2+ from the surrounding solution, and were inactivated in the presence of the effective blocker of TRPV5 and TRPV6, ruthenium red. The current-voltage characteristics of the channels demonstrated an inward rectification. The channel activity in Jurkat cells was significantly higher than in normal HBLs. The real-time RT-PCR analysis revealed a higher level of mRNA of the genes encoding channels TRPV5 and TRPV6 in the proliferating Jurkat T-cells as compared with normal lymphocytes. In general, the data have shown that TRPV5 and TRPV6 channels are expressed in blood lymphocytes are functionally active, and their activity is associated with proliferative status of blood cells.