Increasing evidence indicates that the tumor microenvironment is a critical factor supporting cancer progression, chemoresistance and metastasis. Recently, cancer-associated fibroblasts (CAFs) have been recognized as a crucial tumor stromal component promoting cancer growth and invasiveness via modulation of the extracellular matrix (ECM) structure, tumor metabolism and immune reprogramming. One of the main sources of CAFs are endothelial cells undergoing the endothelial-mesenchymal transition (EndMT). EndMT is mainly promoted by the Transforming Growth Factor-β (TGF-β) family secreted by tumor cells, though the role of particular members in EndMT regulation remains poorly understood. Our findings demonstrate that TGF-β2 induces mesenchymal transdifferentiation of human microvascular endothelial cells (HMEC-1 cells) to CAF-like cells in association with elongated cell morphology, modulation of stress fiber organization, higher α-SMA protein levels and activation of RhoA and Rac-1 pathways. Such regulation is similar to that observed in cells maintained using conditioned medium from invasive colorectal cancer cell line culture. Furthermore, TGF-β2 stimulation resulted in myocardin-related transcription factor (MRTF) activation and upregulation. Our results demonstrate for the first time that such interaction is sufficient for integrin-linked kinase (ILK) overexpression. ILK upregulation also enhanced MRTF activation via RhoA and Rac-1-MMP9 via inside-out integrin activation. Herein, we propose a new ILK-MMP9-MRTF axis that appears to be critical for EndMT differentiation of endothelial to CAF-like cells. Thus, it might be an attractive target for cancer treatment.
Endothelial–mesenchymal transition (EndMT) is a crucial phenomenon in regulating the development of diseases, including cancer metastasis and fibrotic disorders. The primary regulators of disease development are zinc-finger transcription factors belonging to the Snail family. In this study, we characterized the myocardin-related transcription factor (MRTF)-dependent mechanisms of a human snail promoter regulation in TGF-β-stimulated human endothelial cells. Although in silico analysis revealed that the snail promoter’s regulatory fragment contains one GCCG and two SP1 motifs that could be occupied by MRTFs, the genetic study confirmed that MRTF binds only to SP1 sites to promote snail expression. The more accurate studies revealed that MRTF-A binds to both SP1 elements, whereas MRTF-B to only one (SP1near). Although we found that each MRTF alone is capable of inducing snail expression, the direct cooperation of these proteins is required to reinforce snail expression and promote the late stages of EndMT within 48 hours. Furthermore, genetic and biochemical analysis revealed that MRTF-B alone could induce the late stage of EndMT. However, it requires a prolonged time. Therefore, we concluded that MRTFs might cause EndMT in a fast- and slow-dependent manner. Based on MRTF-dependent Snail upregulation, we recognized that TGF-β1, as an MRTF-B regulator, is involved in slow EndMT induction, whereas TGF-β2, which altered both MRTF-A and MRTF-B expression, promotes a fast EndMT process.
Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.
This chapter describes signaling pathways stimulated by the P2Y(2) nucleotide receptor (P2Y(2)R), that regulate cellular processes dependent on actin cytoskeleton dynamics in glioma C6 cells. P2Y(2)R coupled with G-proteins, in response to ATP or UTP, regulates the level of phosphatidylinositol-4,5-bisphosphate (PIP(2)) which modulates a variety of actin binding proteins and is involved in calcium response and activates Rac1 and RhoA proteins. The RhoA/ROCK signaling pathway plays an important role in contractile force generation needed for the assembly of stress fibers, focal adhesions and for tail retraction during cell migration. Blocking of this pathway by a specific Rho-kinase inhibitor induces changes in F-actin organization and cell shape and decreases the level of phosphorylated myosin II and cofilin. In glioma C6 cells these changes are reversed after UTP stimulation of P2Y(2)R. Signaling pathways responsible for this compensation are connected with calcium signaling. Stimulation of the Rac1 mediated pathway via G(o) proteins needs additional interaction between α(v)β(5) integrins and P2Y(2)Rs. Rac1 activation is necessary for cofilin phosphorylation as well as integrin activation needed for focal complexes formation and stabilization of lamellipodium. Inhibition of positive Rac1 regulation prevents glioma C6 cells from recovery of control cell like morphology.
We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid.
Inhibition of Rho-associated protein kinase (ROCK) activity in glioma C6 cells induces changes in actin cytoskeleton organization and cell morphology similar to those observed in other types of cells with inhibited RhoA/ROCK signaling pathway. We show that phosphorylation of myosin light chains (MLC) induced by P2Y₂ receptor stimulation in cells with blocked ROCK correlates in time with actin cytoskeleton reorganization, F-actin redistribution and stress fibers assembly followed by recovery of normal cell morphology. Presented results indicate that myosin light-chain kinase (MLCK) is responsible for the observed phosphorylation of MLC. We also found that the changes induced by P2Y₂ stimulation in actin cytoskeleton dynamics and morphology of cells with inhibited ROCK, but not in the level of phosphorylated MLC, depend on the presence of calcium in the cell environment.
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