RhoGDI1 regulates cell-cell junctions in polarized epithelial cells.

Cell-cell contact formation of polarized epithelial cells is a multi-step process that involves the co-ordinated activities of Rho family small GTPases. Consistent with the central role of Rho GTPases, a number of Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) have been identified at cell-cell junctions at various stages of junction maturation. As opposed to RhoGEFs and RhoGAPs, the role of Rho GDP dissociation inhibitors (GDIs) during cell-cell contact formation is poorly understood. Here, we have analyzed the role of RhoGDI1/ARHGDIA, a member of the RhoGDI family, during cell-cell contact formation of polarized epithelial cells. Depletion of RhoGDI1 delays the development of linear cell-cell junctions and the formation of barrier-forming tight junctions. In addition, RhoGDI1 depletion impairs the ability of cells to stop migration in response to cell collision and increases the migration velocity of collectively migrating cells. We also find that the cell adhesion receptor JAM-A promotes the recruitment of RhoGDI1 to cell-cell contacts. Our findings implicate RhoGDI1 in various processes involving the dynamic reorganization of cell-cell junctions.

Minimal cellular automaton model with heterogeneous cell sizes predicts epithelial colony growth.

Regulation of cell proliferation is a crucial aspect of tissue development and homeostasis and plays a major role in morphogenesis, wound healing, and tumor invasion. A phenomenon of such regulation is contact inhibition, which describes the dramatic slowing of proliferation, cell migration and individual cell growth when multiple cells are in contact with each other. While many physiological, molecular and genetic factors are known, the mechanism of contact inhibition is still not fully understood. In particular, the relevance of cellular signaling due to interfacial contact for contact inhibition is still debated. Cellular automata (CA) have been employed in the past as numerically efficient mathematical models to study the dynamics of cell ensembles, but they are not suitable to explore the origins of contact inhibition as such agent-based models assume fixed cell sizes. We develop a minimal, data-driven model to simulate the dynamics of planar cell cultures by extending a probabilistic CA to incorporate size changes of individual cells during growth and cell division. We successfully apply this model to previous in-vitro experiments on contact inhibition in epithelial tissue: After a systematic calibration of the model parameters to measurements of single-cell dynamics, our CA model quantitatively reproduces independent measurements of emergent, culture-wide features, like colony size, cell density and collective cell migration. In particular, the dynamics of the CA model also exhibit the transition from a low-density confluent regime to a stationary postconfluent regime with a rapid decrease in cell size and motion. This implies that the volume exclusion principle, a mechanical constraint which is the only inter-cellular interaction incorporated in the model, paired with a size-dependent proliferation rate is sufficient to generate the observed contact inhibition. We discuss how our approach enables the introduction of effective bio-mechanical interactions in a CA framework for future studies.

N-cadherin directs the collective Schwann cell migration required for nerve regeneration through Slit2/3-mediated contact inhibition of locomotion.

Collective cell migration is fundamental for the development of organisms and in the adult for tissue regeneration and in pathological conditions such as cancer. Migration as a coherent group requires the maintenance of cell-cell interactions, while contact inhibition of locomotion (CIL), a local repulsive force, can propel the group forward. Here we show that the cell-cell interaction molecule, N-cadherin, regulates both adhesion and repulsion processes during Schwann cell (SC) collective migration, which is required for peripheral nerve regeneration. However, distinct from its role in cell-cell adhesion, the repulsion process is independent of N-cadherin trans-homodimerisation and the associated adherens junction complex. Rather, the extracellular domain of N-cadherin is required to present the repulsive Slit2/Slit3 signal at the cell surface. Inhibiting Slit2/Slit3 signalling inhibits CIL and subsequently collective SC migration, resulting in adherent, nonmigratory cell clusters. Moreover, analysis of ex vivo explants from mice following sciatic nerve injury showed that inhibition of Slit2 decreased SC collective migration and increased clustering of SCs within the nerve bridge. These findings provide insight into how opposing signals can mediate collective cell migration and how CIL pathways are promising targets for inhibiting pathological cell migration.

The Role of Mechanotransduction in Contact Inhibition of Locomotion and Proliferation.

Contact inhibition (CI) represents a crucial tumor-suppressive mechanism responsible for controlling the unbridled growth of cells, thus preventing the formation of cancerous tissues. CI can be further categorized into two distinct yet interrelated components: CI of locomotion (CIL) and CI of proliferation (CIP). These two components of CI have historically been viewed as separate processes, but emerging research suggests that they may be regulated by both distinct and shared pathways. Specifically, recent studies have indicated that both CIP and CIL utilize mechanotransduction pathways, a process that involves cells sensing and responding to mechanical forces. This review article describes the role of mechanotransduction in CI, shedding light on how mechanical forces regulate CIL and CIP. Emphasis is placed on filamin A (FLNA)-mediated mechanotransduction, elucidating how FLNA senses mechanical forces and translates them into crucial biochemical signals that regulate cell locomotion and proliferation. In addition to FLNA, trans-acting factors (TAFs), which are proteins or regulatory RNAs capable of directly or indirectly binding to specific DNA sequences in distant genes to regulate gene expression, emerge as sensitive players in both the mechanotransduction and signaling pathways of CI. This article presents methods for identifying these TAF proteins and profiling the associated changes in chromatin structure, offering valuable insights into CI and other biological functions mediated by mechanotransduction. Finally, it addresses unanswered research questions in these fields and delineates their possible future directions.

A neuroligin-2-YAP axis regulates progression of pancreatic intraepithelial neoplasia.

Pancreatic ductal adenocarcinoma (PDAC) is a tumor with a dismal prognosis that arises from precursor lesions called pancreatic intraepithelial neoplasias (PanINs). Progression from low- to high-grade PanINs is considered as tumor initiation, and a deeper understanding of this switch is needed. Here, we show that synaptic molecule neuroligin-2 (NLGN2) is expressed by pancreatic exocrine cells and plays a crucial role in the regulation of contact inhibition and epithelial polarity, which characterize the switch from low- to high-grade PanIN. NLGN2 localizes to tight junctions in acinar cells, is diffusely distributed in the cytosol in low-grade PanINs and is lost in high-grade PanINs and in a high percentage of advanced PDACs. Mechanistically, NLGN2 is necessary for the formation of the PALS1/PATJ complex, which in turn induces contact inhibition by reducing YAP function. Our results provide novel insights into NLGN2 functions outside the nervous system and can be used to model PanIN progression.

Equations describing semi-confluent cell growth (I) Analytical approximations.

A set of differential equations with analytical solutions are presented that can quantitatively account for variable degrees of contact inhibition on cell growth in two- and three-dimensional cultures. The developed equations can be used for comparative purposes when assessing contribution of higher-order effects, such as culture geometry and nutrient depletion, on mean cell growth rate. These equations also offer experimentalists the opportunity to characterize cell culture experiments using a single reductive parameter.

The phosphodiesterase 2A controls lymphatic junctional maturation via cGMP-dependent notch signaling.

The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.

UBE2A/B is the trans-acting factor mediating mechanotransduction and contact inhibition.

Mechanotransduction and contact inhibition (CI) control gene expression to regulate proliferation, differentiation, and even tumorigenesis of cells. However, their downstream trans-acting factors (TAFs) are not well known due to a lack of a high-throughput method to quantitatively detect them. Here, we developed a method to identify TAFs on the cis-acting sequences that reside in open chromatin or DNaseI-hypersensitive sites (DHSs) and to detect nucleocytoplasmic shuttling TAFs using computational and experimental screening. The DHS-proteomics revealed over 1000 potential mechanosensing TAFs and UBE2A/B (Ubiquitin-conjugating enzyme E2 A) was experimentally identified as a force- and CI-dependent nucleocytoplasmic shuttling TAF. We found that translocation of YAP/TAZ and UBE2A/B are distinctively regulated by inhibition of myosin contraction, actin-polymerization, and CI depending on cell types. Next-generation sequence analysis revealed many downstream genes including YAP are transcriptionally regulated by ubiquitination of histone by UBE2A/B. Our results suggested a YAP-independent mechanotransduction and CI pathway mediated by UBE2A/B.

Autocrine Factors Produced by Mesenchymal Stem Cells in Response to Cell-Cell Contact Inhibition Have Anti-Tumor Properties.

Recently, mesenchymal stem cell (MSC) therapies have been questioned as MSCs are capable of both promoting and inhibiting tumorigenesis. Both MSCs and tumor cells replicate to increase their population size; however, MSCs, but not tumor cells, stop dividing when they reach confluence due to cell-cell contact inhibition and then differentiate. We hypothesized that contact inhibition results in the production of effector molecules by confluent MSCs and these effectors are capable of suppressing tumor cell growth. To test this hypothesis, we co-cultured breast cancer cells (MDA-MB-231) with either confluent or sub-confluent bone-marrow-derived MSCs (BM-MSCs); in addition, we treated various tumor cells with conditioned media (CM) obtained from either confluent or sub-confluent BM-MSCs. The results showed that the growth of tumor cells co-cultured with confluent BM-MSCs or treated with CM obtained from confluent BM-MSCs was inhibited, and this effect was significantly stronger than that seen with tumor cells co-cultured with sub-confluent BM-MSCs or CM obtained from sub-confluent BM-MSCs. Subcutaneous tumor formation was completely prevented by the inoculation of tumor cells mixed with CM. In the future, soluble anti-tumor effectors, produced by confluent MSCs, may be used as cell-free therapeutics; this approach provides a solution to current concerns associated with cell-based therapies.

Tumour follower cells: A novel driver of leader cells in collective invasion (Review).

Collective cellular invasion in malignant tumours is typically characterized by the cooperative migration of multiple cells in close proximity to each other. Follower cells are led away from the tumour by specialized leader cells, and both cell populations play a crucial role in collective invasion. Follower cells form the main body of the migration system and depend on intercellular contact for migration, whereas leader cells indicate the direction for the entire cell population. Although collective invasion can occur in epithelial and non­epithelial malignant neoplasms, such as medulloblastoma and rhabdomyosarcoma, the present review mainly provided an extensive analysis of epithelial tumours. In the present review, the cooperative mechanisms of contact inhibition locomotion between follower and leader cells, where follower cells coordinate and direct collective movement through physical (mechanical) and chemical (signalling) interactions, is summarised. In addition, the molecular mechanisms of follower cell invasion and metastasis during remodelling and degradation of the extracellular matrix and how chemotaxis and lateral inhibition mediate follower cell behaviour were analysed. It was also demonstrated that follower cells exhibit genetic and metabolic heterogeneity during invasion, unlike leader cells.

Knockout of all ErbB-family genes delineates their roles in proliferation, survival and migration.

The ErbB-family receptors play pivotal roles in the proliferation, migration and survival of epithelial cells. Because our knowledge on the ErbB-family receptors has been largely obtained by the exogenous application of their ligands, it remains unknown to what extent each of the ErbB members contributes to these outputs. We here knocked out each ErbB gene, various combinations of ErbB genes or all ErbB genes in Madin-Darby canine kidney cells to delineate the contribution of each gene. ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) activation waves during collective cell migration were mediated primarily by ErbB1 and secondarily by the ErbB2 and ErbB3 heterodimer. Either ErbB1 or the ErbB2 and ErbB3 complex was sufficient for the G1/S progression. The saturation cell density was markedly reduced in cells deficient in all ErbB proteins, but not in cells retaining only ErbB2, which cannot bind to ligands. Thus, a ligand-independent ErbB2 activity is sufficient for preventing apoptosis at high cell density. In short, systematic knockout of ErbB-family genes has delineated the roles of each ErbB receptor.

The Tumor Suppressor DAB2IP Is Regulated by Cell Contact and Contributes to YAP/TAZ Inhibition in Confluent Cells.

External and internal mechanical forces modulate cell morphology, movement, proliferation and metabolism, and represent crucial inputs for tissue homeostasis. The transcriptional regulators YAP and TAZ are important effectors of mechanical signaling and are frequently activated in solid tumors, correlating with metastasis, chemoresistance, and shorter patient survival. YAP/TAZ activity is controlled by various pathways that sense cell shape, polarity, contacts, and mechanical tension. In tumors, aberrant YAP/TAZ activation may result from cancer-related alterations of such regulatory networks. The tumor suppressor DAB2IP is a Ras-GAP and scaffold protein that negatively modulates multiple oncogenic pathways and is frequently downregulated or inactivated in solid tumors. Here, we provide evidence that DAB2IP expression is sustained by cell confluency. We also find that DAB2IP depletion in confluent cells alters their morphology, reducing cell packing while increasing cell stiffness. Finally, we find that DAB2IP depletion in confluent cells favors YAP/TAZ nuclear localization and transcriptional activity, while its ectopic expression in subconfluent cells increases YAP/TAZ retention in the cytoplasm. Together, these data suggest that DAB2IP may function as a sensor of cell interactions, contributing to dampening cellular responses to oncogenic inputs in confluent cells and that DAB2IP loss-of-function would facilitate YAP/TAZ activation in intact epithelia, accelerating oncogenic transformation.

An E-cadherin-actin clutch translates the mechanical force of cortical flow for cell-cell contact to inhibit epithelial cell locomotion.

Adherens junctions (AJs) allow cell contact to inhibit epithelial migration yet also permit epithelia to move as coherent sheets. How, then, do cells identify which contacts will inhibit locomotion? Here, we show that in human epithelial cells this arises from the orientation of cortical flows at AJs. When the leader cells from different migrating sheets make head-on contact with one another, they assemble AJs that couple together oppositely directed cortical flows. This applies a tensile signal to the actin-binding domain (ABD) of α-catenin, which provides a clutch to promote lateral adhesion growth and inhibit the lamellipodial activity necessary for migration. In contrast, AJs found between leader cells in the same migrating sheet have cortical flows aligned in the same direction, and no such mechanical inhibition takes place. Therefore, α-catenin mechanosensitivity in the clutch between E-cadherin and cortical F-actin allows cells to interpret the direction of motion via cortical flows and signal for contact to inhibit locomotion.

Epithelial tissue confinement inhibits cell growth and leads to volume-reducing divisions.

Cell proliferation is a central process in tissue development, homeostasis, and disease, yet how proliferation is regulated in the tissue context remains poorly understood. Here, we introduce a quantitative framework to elucidate how tissue growth dynamics regulate cell proliferation. Using MDCK epithelial monolayers, we show that a limiting rate of tissue expansion creates confinement that suppresses cell growth; however, this confinement does not directly affect the cell cycle. This leads to uncoupling between rates of cell growth and division in epithelia and, thereby, reduces cell volume. Division becomes arrested at a minimal cell volume, which is consistent across diverse epithelia in vivo. Here, the nucleus approaches the minimum volume capable of packaging the genome. Loss of cyclin D1-dependent cell-volume regulation results in an abnormally high nuclear-to-cytoplasmic volume ratio and DNA damage. Overall, we demonstrate how epithelial proliferation is regulated by the interplay between tissue confinement and cell-volume regulation.

Distinctions in bone matrix nanostructure, composition, and formation between osteoblast-like cells, MG-63, and human mesenchymal stem cells, UE7T-13.

Osteoblast-like cells and human mesenchymal stem cells (hMSCs) are frequently employed as osteoprogenitor cell models for evaluating novel biomaterials in bone healing and tissue engineering. In this study, the characterization of UE7T-13 hMSCs and MG-63 human osteoblast-like cells was examined. Both cells can undergo osteogenesis and produce calcium extracellular matrix; however, calcium nodules produced by MG-63 lacked a central mass and appeared flatter than UE7T-13. The absence of growing calcium nodules in MG-63 was discovered by SEM-EDX to be associated with the formation of alternating layers of cells and calcium extracellular matrix. The nanostructure and composition analysis showed that UE7T-13 had a finer nanostructure of calcium nodules with a higher calcium/phosphate ratio than MG-63. Both cells expressed high intrinsic levels of collagen type I alpha 1 chain, while only UE7T-13 expressed high levels of alkaline phosphatase, biomineralization associated (ALPL). High ALP activity in UE7T-13 was not further enhanced by osteogenic induction, but in MG-63, low intrinsic ALP activity was greatly induced by osteogenic induction. These findings highlight the differences between the two immortal osteoprogenitor cell lines, along with some technical notes that should be considered while selecting and interpreting the pertinent in vitro model.

Emergence of Spatial Scales and Macroscopic Tissue Dynamics in Active Epithelial Monolayers.

Migrating cells in tissues are often known to exhibit collective swirling movements. In this paper, we develop an active vertex model with polarity dynamics based on contact inhibition of locomotion (CIL). We show that under this dynamics, the cells form steady-state vortices in velocity, polarity, and cell stress with length scales that depend on polarity alignment rate (ζ), self-motility (v0), and cell-cell bond tension (λ). When the ratio λ/v0 becomes larger, the tissue reaches a near jamming state because of the inability of the cells to exchange their neighbors, and the length scale associated with tissue kinematics increases. A deeper examination of this jammed state provides insights into the mechanism of sustained swirl formation under CIL rule that is governed by the feedback between cell polarities and deformations. To gain additional understanding of how active forcing governed by CIL dynamics leads to large-scale tissue dynamics, we systematically coarse-grain cell stress, polarity, and motility and show that the tissue remains polar even on larger length scales. Overall, we explore the origin of swirling patterns during collective cell migration and obtain a connection between cell-level dynamics and large-scale cellular flow patterns observed in epithelial monolayers.

Human umbilical cord mesenchymal stem cells conditioned medium exerts anti-tumor effects on KGN cells in a cell density-dependent manner through activation of the Hippo pathway.

BACKGROUND: The conditioned medium from human umbilical cord mesenchymal stem cells (UCMSCs-CM) provides a new cell-free therapy for tumors due to its unique secretome. However, there are many contradictory reports about the effect of UCMSCs-CM on tumor cells. The loss of contact inhibition is a common characteristic of tumor cells. A relationship between the effect of UCMSCs-CM on tumor cells and contact inhibition in tumor cells is rarely concerned. Whether the effect of UCMSCs-CM on tumor cells is affected by cell density? Here, we explored the effect of UCMSCs-CM on granulosa tumor cell line (KGN) cells at low or high density. METHODS: Growth curve and CCK8 assay were used to assess cell proliferation and viability. Scratch wound and matrigel invasion assay were implicated to detect cell motility of KGN cells. UCMSCs-CM effects on cell cycle, apoptosis and pathway-related proteins were investigated by flow cytometry, TUNEL assay, western blot and immunofluorescence analysis respectively. RESULTS: In growth curve analysis, before KGN cells proliferated into confluence, UCMSCs-CM had no effect on cell proliferation. However, once the cells proliferate to contact each other, UCMSCs-CM significantly inhibited proliferation. Meanwhile, when KGN cells were implanted at high density, UCMSCs-CM could induce cell cycle arrest at G1 phase, inhibit cell migration, invasion and promote apoptosis. While it had no similar effect on KGN cells implanted at low density. In mechanism, the UCMSCs-CM treatment activated the Hippo pathway when KGN cells were implanted at high density. Consistently, the MST1/2 inhibitor, XMU-MP-1, inhibited the activation of the Hippo pathway induced by UCMSCs-CM treatment and accordingly declined the anti-tumor effect of UCMSCs-CM on KGN cells. CONCLUSIONS: The effect of UCMSCs-CM on tumor cells is affected by cell density. UCMSCs-CM exerted anti-tumor effect on KGN cells by activating Hippo pathway to restore contact inhibition. Our results suggest that UCMSCs-CM is a promising therapeutic candidate for GCT treatment.

The Complex Roles of DNA Repair Pathways, Inhibitors, Hyperthermia, and Contact Inhibition in Cell Cycle Halts.

The cell cycle has the capacity to safeguard the cell's DNA from damage. Thus, cell cycle arrest can allow tumor cells to investigate their own DNA repair processes. Cancer cells become extremely reliant on G1-phase cyclin-dependent kinases due to mutated oncogenes and deactivated tumor suppressors, producing replication stress and DNA damage during the S phase and destroying checkpoints that facilitate progression through the S/G2/M phase. DNA damage checkpoints activate DNA repair pathways to prevent cell proliferation, which occurs when the genome is damaged. However, research on how cells recommence division after a DNA lesion-induced arrest is insufficient which is merely the result of cancer cells' susceptibility to cell cycle arrest. For example, defects in the G1 arrest checkpoint may cause a cancer cell to proliferate more aggressively, and attempts to fix these complications may cause the cell to grow more slowly and eventually die. Defects in the G2-M arrest checkpoint may enable a damaged cell to enter mitosis and suffer apoptosis, and attempts to boost the effectiveness of chemotherapy may increase its cytotoxicity. Alternatively, attempts to promote G2-M arrest have also been linked to increased apoptosis in the laboratory. Furthermore, variables, such as hyperthermia, contact inhibition, nucleotide shortage, mitotic spindle damage, and resting phase effects, and DNA replication inhibitors add together to halt the cell cycle. In this review, we look at how nucleotide excision repair, MMR, and other variables, such as DNA replication inhibitors, hyperthermia, and contact inhibition, contribute to the outlined processes and functional capacities that cause cell cycle arrest.

Contact Inhibition of Proliferation Is Accompanied by Expression of the PHF10D Subunit of the Chromatin Remodeling Complex PBAF in Mouse and Human Cell Lines.

PHF10 is a subunit of the PBAF complex, which regulates the expression of many genes in developing and maturing organisms. PHF10 has four isoforms that differ in domain structure. The PHF10A isoform, containing a DPF domain at the C-terminus and 46 amino acids at the N-terminus, is necessary for the expression of proliferation genes; the functions of the other isoforms are less studied. In this work, we have established that, upon contact inhibition of mouse and human cell proliferation caused by the establishment of a tight junction and adherence junction between cells, the expression of the PHF10A isoform stops and instead the PHF10D isoform is expressed, which does not contain DPF-domain and N-terminal sequence. The function of the PHF10D isoform may be associated with the establishment of intercellular contacts.