Downregulation of TRPV6 channel activity by cholesterol depletion in Jurkat T cell line.

Transient receptor potential vanilloid 6 (TRPV6) channels are key players in calcium metabolism of healthy and cancerous cells. Nevertheless, the mechanisms controlling abundance of these channels in plasma membrane of the cells to regulate Ca2+ transport is still poorly understood. In this study, we provide the first evidence that TRPV6 calcium channels and Ca 2+ influx in Jurkat T cell line are modulated by cholesterol, a main lipid component of the plasma membrane. Using patch-clamp technique, we found that activity of TRPV6 channels decreased by cholesterol sequestration with methyl-ß-cyclodextrin (MßCD). Continuous measurement of intracellular Ca2+ revealed a reduction of Ca2+ influx into Jurkat cells following cholesterol depletion. Immunofluorescence and immunoelectron microscopy analyses of MßCD-treated cells detected the lower surface expression of the TRPV6 proteins in comparison with control cells. In general, our data showed that cholesterol regulates TRPV6 channel activity and TRPV6-mediated Ca2+ influx in cells, apparently affecting the localization and density of the calcium channels in the plasma membrane of Jurkat T cells.

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.

Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1.

It has been previously reported that N-terminus of mutant huntingtin (product of the 1st exon) is sufficient to cause a Huntington's disease (HD) pathological phenotype. In view of recent data suggesting that improper regulation of store-operated calcium (SOC) channels is involved in neurodegenerative processes, we investigated influence of expression of the mutant huntingtin N-terminal fragment (Htt138Q-1exon) on SOC entry (SOCE) in mouse neuroblastoma cells (Neuro-2a) and in primary culture of medium spiny neurons (MSNs) isolated from mice. The results show that SOCE in these cells is enhanced upon lentiviral expression of the Htt138Q-1exon. Moreover, we demonstrated that RNAi-mediated knockdown of TRPC1, Orai1, or STIM1 proteins leads to dramatic reduction of abnormal SOCE in both Neuro-2a and MSNs, expressing Htt138Q-1exon. Thus, we concluded that abnormal SOCE in these cells is maintained by both TRPC1- and Orai1-containing channels and required STIM1 for its activation. Furthermore, EVP4593 compound previously tested as a potential anti-HD drug in a Drosophila screening system has proved to be capable of reducing SOCE to the normal level in MSNs expressing the Htt138Q-1exon.

Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells.

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated ß-galactosidase (SA-ß-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny.

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.

Induction of Wnt/ß-catenin signaling in mouse mesenchymal stem cells is associated with activation of the p130 and E2f4 and formation of the p130/Gsk3ß/ß-catenin complex.

Proteins p130 and E2f4, members of the retinoblastoma protein (pRb) family/E2F transcription factor family, are the key elements in regulation of cell cycle and differentiation. The functional role of the p130/E2f4 in mesenchymal stem cells (MSC) is unclear. We demonstrate here that activation of the Wnt/ß-catenin pathway in mouse MSC is associated with accumulation of active forms of the p130, E2f4, and ß-catenin but does not result in inhibition of cell cycle progression. The levels and phosphorylation patterns of p130, E2f4, and ß-catenin in MSC do not change during cell cycle progression. This is different from control T98G glyoblastoma cells that accumulated differently phosphorylated forms of the p130 in quiescence, and under active proliferation. In MSC, synchronized at G0/G1 and S cell cycle phases, the p130 and ß-catenin physically interact each other, whereas Gsk3ß was associated and co-precipitated with both p130 and ß-catenin. Our results indicate that Wnt/ß-catenin and pRb signal pathways interact with each other and form common p130/Gsk3ß/ß-catenin complex during MSC cycle progression. Physiological relevance of such complex may be associated with coupling of the cell cycle and differentiation in MSC, which is related to a wide differentiation potential of these stem cells.

EGF-induced apoptosis in A431 cells is dependent on STAT1, but not on STAT3.

EGF in high concentrations has a growth-inhibitory effect on human epidermoid carcinoma cells A431. The transcription factor STAT1 is the most probable candidate for mediating this effect. In the present study, we demonstrated a strong reduction of the expression level of STAT1 in EGF-resistant sub-clones of A431 cells. EGF resistance was reversed by introducing wild-type STAT1, but not its Y701F mutant. Moreover, blocking the activity of Src family kinases reduced tyrosine phosphorylation of STAT1 and STAT3 and protected A431 cells from the EGF-induced growth inhibition. To further elucidate roles of STATs in A431 cell growth and survival, clones of A431 cells expressing short hairpin RNA (shRNA) against STAT1 or STAT3 were generated. Neither STAT1 nor STAT3 knockdown exerted any effect on growth rate or apoptotic death of A431 cells in the absence of EGF. However, upon EGF treatment A431 cells with knocked down STAT1 continued to grow and demonstrated a significantly lower level of apoptosis as compared to A431 cells. The knockdown of STAT3 did not alter cell growth or apoptosis. Taken together, our experiments prove the essential role of tyrosine phosphorylated STAT1, but not of STAT3, in EGF-induced apoptosis in A431 cells.