Chondrogenic differentiation followed IGFBP3 loss in human endometrial mesenchymal stem cells.

Insulin-like growth factor binding protein 3 (IGFBP3) is a multifunctional protein, able either to stimulate the cell growth or to promote apoptosis. In particular, IGFBP3 plays significant role in propagation of stress-induced senescence in human endometrium-derived mesenchymal stem cells (MESCs) (Vassilieva et al., 2020). We undertook CRISPR/Cas9-mediated IGFBP3 knockout in an effort to decelerate stress-induced senescence in MESCs, but, unexpectedly, IGFBP3-knockout MESCs culture acquired chondrocyte-like features, such as cell condensation and aggregation. We revealed that IGFBP3-knockout MESCs completely lost CD73 and CD90 MESCs positive surface markers, and significantly decreased expression of CD105 and CD146 MESCs positive surface markers. In addition, we found IGFBP3-knockout MESCs aggregates positively stained for Alcian Blue. We also detected expression of collagen type II in IGFBP3-knockout MESCs. The obtained results indicate that MESCs lost stemness after IGFBP3-knockout and underwent differentiation toward chondrogenic lineage. Our findings can enlighten IGFBP3 role in regulation of MESCs chondrogenesis.

Agonist-induced Piezo1 activation suppresses migration of transformed fibroblasts.

Increased migratory, invasive and metastatic potential is one of the main pathophysiological determinants of malignant cells. Mechanosensitive calcium-permeable ion channels are among the key membrane proteins that participate in processes of cellular motility. Local calcium influx via mechanosensitive channels was proposed to regulate calcium-dependent molecules involved in cell migration. Piezo transmembrane proteins were shown to act as calcium-permeable mechanosensitive ion channels in various cells and tissues, including a number of tumor cells. Furthermore, an elevated expression of Piezo1 is correlated with poor prognosis for some types of cancers. At the same time, functional impact of Piezo1 channels on pathophysiological reactions of tumor cells remains largely unknown. Here, we used 3T3B-SV40 mouse fibroblasts as a model to study the effect of Yoda1, selective Piezo1 activator, on migrative properties of transformed cells. RT-PCR and immunofluorescent staining showed the presence of native Piezo1 in 3T3B-SV40 fibroblasts. Functional expression of Piezo1 in plasma membrane of 3T3B-SV40 cells was confirmed by calcium measurements and single channel patch-clamp analysis. Particularly, application of Yoda1 resulted in rapid calcium influx and induced typical channel activity in membrane patches with characteristics identical to stretch-activated channels in 3T3B-SV40 cells. Importantly, dose-dependent inhibition of cellular migration by Yoda1 was found in wound healing assay using live cell imaging. Consistently, microscopic analysis showed that Yoda1 significantly altered cellular morphology, induced F-actin assembly and stress fiber formation indicating partial reversion of transformed phenotype. The results demonstrate for the first time that Piezo1 activation by selective agonist Yoda1 could be favorable for inhibiting migrative potential of transformed cells with native Piezo1 expression.

Extracellular protease trypsin activates amiloride-insensitive sodium channels in human leukemia cells.

Sodium influx is tightly regulated in the cells of blood origin. Amiloride-insensitive sodium channels were identified as one of the main sodium-transporting pathways in leukemia cells. To date, all known regulatory pathways of these channels are coupled with intracellular actin cytoskeleton dynamics. Here, to search for physiological mechanisms controlling epithelial Na+ channel (ENaC)-like channels, we utilized leukemia K562 cells as a unique model to examine single channel behavior in a whole-cell patch-clamp experiments. We have shown for the first time that extracellular serine protease trypsin directly activates sodium channels in plasma membrane of K562 cells. The whole-cell single current recordings clearly demonstrate no inhibition of trypsin-activated channels by amiloride or benzamil. Involvement of proteolytic cleavage in channel opening was confirmed in experiments with soybean trypsin inhibitor. More importantly, stabilization of F-actin with intracellular phalloidin did not prevent trypsin-induced channel activation indicating no implication of cytoskeleton rearrangements in stimulatory effect of extracellular protease. Our data reveals a novel mechanism modulating amiloride-insensitive ENaC-like channel activity and integral sodium permeability in leukemia cells.

Senescence-messaging secretome factors trigger premature senescence in human endometrium-derived stem cells.

Accumulating evidence suggests that the senescence-messaging secretome (SMS) factors released by senescent cells play a key role in cellular senescence and physiological aging. Phenomenon of the senescence induction in human endometrium-derived mesenchymal stem cells (MESCs) in response to SMS factors has not yet been described. In present study, we examine a hypothesis whether the conditioned medium from senescent cells (CM-old) may promote premature senescence of young MESCs. In this case, we assume that SMS factors, containing in CM-old are capable to trigger senescence mechanism in a paracrine manner. A long-term cultivation MESCs in the presence of CM-old caused deceleration of cell proliferation along with emerging senescence phenotype, including increase in both the cell size and SA-ß-Gal activity. The phosphorylation of p53 and MAPKAPK-2, a direct target of p38MAPK, as well as the expression of p21Cip1 and p16Ink4a were increased in CM-old treated cells with senescence developing whereas the Rb phosphorylation was diminished. The senescence progression was accompanied by both enhanced ROS generation and persistent activation of DNA damage response, comprising protein kinase ATM, histone H2A.X, and adapter protein 53BP1. Thus, we suggest that a senescence inducing signal is transmitted through p16/MAPKAPK-2/Rb and DDR-mediated p53/p21/Rb signaling pathways. This study is the first to demonstrate that the SMS factors secreted in conditioned medium of senescent MESCs trigger a paracrine mechanism of premature senescence in young cells.

Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells.

Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances in hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca2+ entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem 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.

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

Regulation of Ca(2+) 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 [Ca(2+)](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 Ca(2+)-selective members of the TRPs, Ca(2+) 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 Ca(2+) 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.

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.

Thymocyte K+, Na+ and water balance during dexamethasone- and etoposide-induced apoptosis.

The mechanism of apoptotic cell volume decrease was studied in rat thymocytes treated with dexamethasone (Dex) or etoposide (Eto). Cell shrinkage, i.e. dehydration, was quantified by using buoyant density of the thymocytes in a continuous Percoll gradient. The K+ and Na+ content of cells from different density fractions were assayed by flame emission analysis. Apoptosis was tested by microscopy and flow cytometry of acridine orange stained cells as well as by flow DNA cytometry. Treatment of the thymocytes with 1 microM Dex for 4-5.5 h or 50 microM Eto for 5 h resulted in the appearance of a new distinct high-density cell subpopulation. The cells from this heavy subpopulation but not those with normal buoyant density had typical features of apoptosis. Apoptotic increase of cell density was accompanied by a decrease in cellular K+ content, which exceeded the simultaneous increase in cellular Na+ content. Cellular loss of K+ contributed to most of the estimated loss of cellular osmolytes, but owing to the parallel loss of cell water, the decrease in cytosolic K+ concentration was less than one third. Due to gain of Na+ and loss of cell water the cytosolic Na+ concentration in thymocytes rose following treatment with Dex (5.5 h) or Eto (5 h) by a factor of about 3.6 and 3.1, respectively.

Properties of Mg(2+)-dependent cation channels in human leukemia K562 cells.

The endogenous Mg(2+)-inhibited cation (MIC) current was recently described in different cells of hematopoietic lineage and was implicated in the regulation of Mg2+ homeostasis. Here we present a single channel study of endogenously expressed Mg(2+)-dependent cation channels in the human myeloid leukemia K562 cells. Inwardly directed unitary currents were activated in cell-attached experiments in the absence of Ca2+ and Mg2+ in the pipette solution. The current-voltage (I-V) relationships displayed strong inward rectification and yielded a single channel slope conductance of approximately 30 pS at negative potentials. The I-V relationships were not altered by patch excision into divalent-free solution. Channel open probability (P(o)) and mean closed time constant (tau(C)) were strongly voltage-dependent, indicating that gating mechanisms may underlie current inward rectification. Millimolar concentrations of Ca2+ or Mg2+ applied to the cytoplasmic side of the membrane produced slow irreversible inhibition of channel activity. The Mg(2+)-dependent cation channels described in this study differ from the MIC channels described in human T-cells, Jurkat, and rat basophilic leukemia (RBL) cells in their I-V relationships, kinetic parameters and dependence on intracellular divalent cations. Our results suggested that endogenously expressed Mg(2+)-dependent cation channels in K562 cells and the MIC channels in other hematopoietic cells might be formed by different channel proteins.