Direct Cell-cell Communication Research Articles

The components of an electrical synapse as revealed by expansion microscopy of a single synaptic contact.

Most nervous systems combine both transmitter-mediated and direct cell-cell communication, known as 'chemical' and 'electrical' synapses, respectively. Chemical synapses can be identified by their multiple structural components. Electrical synapses are, on the other hand, generally defined by the presence of a 'gap junction' (a cluster of intercellular channels) between two neuronal processes. However, while gap junctions provide the communicating mechanism, it is unknown whether electrical transmission requires the contribution of additional cellular structures. We investigated this question at identifiable single synaptic contacts on the zebrafish Mauthner cells, at which gap junctions coexist with specializations for neurotransmitter release and where the contact unequivocally defines the anatomical limits of a synapse. Expansion microscopy of these single contacts revealed a detailed map of the incidence and spatial distribution of proteins pertaining to various synaptic structures. Multiple gap junctions of variable size were identified by the presence of their molecular components. Remarkably, most of the synaptic contact's surface was occupied by interleaving gap junctions and components of adherens junctions, suggesting a close functional association between these two structures. In contrast, glutamate receptors were confined to small peripheral portions of the contact, indicating that most of the synaptic area functions as an electrical synapse. Thus, our results revealed the overarching organization of an electrical synapse that operates with not one, but multiple gap junctions, in close association with structural and signaling molecules known to be components of adherens junctions. The relationship between these intercellular structures will aid in establishing the boundaries of electrical synapses found throughout animal connectomes and provide insight into the structural organization and functional diversity of electrical synapses.

SNP and Structural Study of the Notch Superfamily Provides Insights and Novel Pharmacological Targets against the CADASIL Syndrome and Neurodegenerative Diseases.

The evolutionary conserved Notch signaling pathway functions as a mediator of direct cell-cell communication between neighboring cells during development. Notch plays a crucial role in various fundamental biological processes in a wide range of tissues. Accordingly, the aberrant signaling of this pathway underlies multiple genetic pathologies such as developmental syndromes, congenital disorders, neurodegenerative diseases, and cancer. Over the last two decades, significant data have shown that the Notch signaling pathway displays a significant function in the mature brains of vertebrates and invertebrates beyond neuronal development and specification during embryonic development. Neuronal connection, synaptic plasticity, learning, and memory appear to be regulated by this pathway. Specific mutations in human Notch family proteins have been linked to several neurodegenerative diseases including Alzheimer's disease, CADASIL, and ischemic injury. Neurodegenerative diseases are incurable disorders of the central nervous system that cause the progressive degeneration and/or death of brain nerve cells, affecting both mental function and movement (ataxia). There is currently a lot of study being conducted to better understand the molecular mechanisms by which Notch plays an essential role in the mature brain. In this study, an in silico analysis of polymorphisms and mutations in human Notch family members that lead to neurodegenerative diseases was performed in order to investigate the correlations among Notch family proteins and neurodegenerative diseases. Particular emphasis was placed on the study of mutations in the Notch3 protein and the structure analysis of the mutant Notch3 protein that leads to the manifestation of the CADASIL syndrome in order to spot possible conserved mutations and interpret the effect of these mutations in the Notch3 protein structure. Conserved mutations of cysteine residues may be candidate pharmacological targets for the potential therapy of CADASIL syndrome.

Unraveling and Visualizing the Interactions of Acute Myeloid Leukemia with the Bone Marrow Microenvironment in Vitro and In Vivo

The bone marrow (BM) is a dynamic stem cell microenvironment, harboring hematopoietic stem cells (HSCs) and various non-blood components such as endothelium, mural cells, multipotent stromal cells (MSCs) and their derivatives including osteoblasts, adipocytes, and fibroblasts. This stromal niche supports HSC-fate decisions and has been proposed as a major determinant of chemoresistance in hematological malignancies, such as acute myeloid leukemia (AML). Following chemotherapy, AML patients often suffer from relapse, in part due to chemoresistant leukemic stem cells (LSCs). There have been various indications in literature that leukemic cells remodel their niche, creating a permissive microenvironment for LSCs. However, which cell types respond and which underlying molecular mechanisms are involved skewing the compositional changes in the BM microenvironment remain unclear. Here, we aim to investigate how high LSC frequency (LSChigh) versus low LSC frequency (LSClow) burden AML initiates BM niche remodeling in the favor of AML expansion, with a particular focus on the MSC lineages. We further aim to determine which stromal cell types interact with LSCs and how their composition is altered upon AML development. Additionally, we aim to visualize which cell types interact with and are positioned in close proximity to LSCs. We combine single-cell transcriptomics of AML xenograft models and in vitro co-cultures of MSCs with primary AML. First, we set up clinically relevant murine xenograft models in which primary AML cells with either a low vs high LSC frequency (Garg et al., 2019) were injected in immune-deficient NSGW41 mice. As control, we injected cord blood-derived HSPCs. 6-7 weeks after engraftment, we harvested and digested the bones using our established protocol (adapted from Baccin et al., 2020) to recover the stromal cells with high efficiency. Besides, we also kept a femur per mouse for follow-up (multiplexed) immunofluorescence imaging. We enriched for human and mouse HSPCs, human AML cells, and mouse niche cells using FACS-based sorting, followed by scRNA-seq. Additionally, we isolated BM-derived mononuclear cells from healthy donors and AML patients and expanded the MSCs in vitro. We set up 4 independent co-cultures with MSCs and primary AML cells with LSChigh versus LSClow frequency. After 48 hours, we performed scRNA-seq to investigate transcriptomic changes and subsequent variant biological processes in the MSCs. scRNA-seq profiling of the BM niche of our AML xenograft models demonstrates that AML presence significantly impacts the niche composition, leading to the expansion of Cxcl12-abundant-reticular adipocytes (adipo-CAR) and the decline of osteoblasts. When comparing LSChigh versus LSClow, we detect significant differences in abundance of certain stromal cells, including endothelium, adipo-CARs, and fibroblast subtypes. This suggests that the LSC-burden plays a critical role in the extent of niche remodeling. To further uncover the direct effects of LSCs on MSCs, we have established an AML-MSC co-culture system and perform scRNA-seq on the MSCs. In line with our in vivo data, we observe that cultured BM MSCs, especially upon exposure to LSChigh AML, suppress osteoblast lineage genes while overexpressing fibrosis-related genes. We are validating our observations using multiplexed immunofluorescence to further investigate the direct cell-cell communication of LSCs with their microenvironment. Taken together, our data in murine AML models and human AML-MSC co-cultures unravel new insights into the BM stem cell niche composition and highlight a specific LSC-induced skewing of the mesenchymal lineage. Our data helps to better understand to which extent the BM microenvironment impacts AML disease progression and relapse due to residual LSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Connexin-Based Channels in the Liver.

Connexin proteins oligomerize in hexameric structures called connexin hemichannels, which then dock to form gap junctions. Gap junctions direct cell-cell communication by allowing the exchange of small molecules and ions between neighboring cells. In this way, hepatic gap junctions support liver homeostasis. Besides serving as building blocks for gap junctions, connexin hemichannels provide a pathway between the intracellular and the extracellular environment. The activation of connexin hemichannels is associated with acute and chronic liver pathologies. This article discusses the role of gap junctions and connexin hemichannels in the liver. © 2022 American Physiological Society. Compr Physiol 12:1-17, 2022.

P694: INVESTIGATING THE ROLE OF CELL ADHESION MOLECULES IN TUNNELING NANOTUBES FORMATION IN CHRONIC MYELOID LEUKEMIA MICROENVIRONMENT

Background: The cross-talk and interactions between leukemia cells and their microenvironment play an important role in the development of drug resistance. Tunneling nanotubes (TNTs) represent a novel type of direct cell-cell communication way allowing for direct transfer of different types of cargo between distant cells. Our previous studies (Kolba et al., 2019) have shown that these structures are formed between bone marrow stromal HS-5 cells and chronic myeloid leukemia (CML) K562 cells. Significantly, TNT-mediated transfer of membrane vesicles from stromal to leukemic cells resulted in the stroma-mediated protection of CML cells from imatinib-induced apoptosis. Despite the efforts made to understand the molecular mechanisms regulating TNTs formation, they still remain unclear - especially in the leukemia microenvironment. We have shown that TNTs are formed between stromal and leukemic cells, following direct cell-cell interaction and cell dislodgement. Thus, we have focused our attention on cell adhesion molecules, as potential key players in tunneling nanotubes formation and TNT-mediated vesicles transfer between leukemic and stromal cells. Aims: The aim of presented studies was to elucidate the role of CD44 and ITGβ1 - one of the most important cell adhesion molecules, in TNTs formation between leukemic and stromal cells and TNT-mediated exchange of vesicles. Methods: To examine this, we established the HS-5 stromal cell line with silenced CD44 protein expression. Then, we co-cultured control (wt) or CD44-silenced HS-5 cells with leukemic K562 cells. For ITGβ1 studies, we used blocking anti-ITGβ1 antibody. Using confocal microscopy, three types of TNTs in co-cultures were counted: homotypic TNTs formed between stromal cells, homotypic TNTs formed between CML cells and TNTs formed between stromal and CML cells (heterotypic). Moreover, the TNT-mediated transfer of cellular vesicles from stromal to leukemic cells was assessed in these co-cultures using flow cytometry. Since CD44 expression increases in CML cells after co-culture with stroma, we examined its potential role in the drug resistance in CML acceptor cells. To do this, rhodamine-123 efflux assay was used. Results: We found that both, tunneling nanotubes number and uptake of cellular vesicles by leukemia, decreased after co-culture with CD44-silenced stromal cells and after blocking with anti-ITGβ1 antibody. Flow cytometry analyses of rhodamine-123 efflux showed significantly increased efflux pump activity in leukemic acceptor cells. Summary/Conclusion: Our data suggest that CD44 and ITGβ1 can play an important role in formation of tunneling nanotubes between leukemic and stromal cells and TNT-mediated cellular vesicles exchange. Moreover, CD44 presented in acceptor cells is involved in the development of drug resistance in leukemic cells, by promoting activity of efflux transporters.

Direct Cell-Cell Communication via Membrane Pores, Gap Junction Channels, and Tunneling Nanotubes: Medical Relevance of Mitochondrial Exchange.

The history of direct cell-cell communication has evolved in several small steps. First discovered in the 1930s in invertebrate nervous systems, it was thought at first to be an exception to the “cell theory”, restricted to invertebrates. Surprisingly, however, in the 1950s, electrical cell-cell communication was also reported in vertebrates. Once more, it was thought to be an exception restricted to excitable cells. In contrast, in the mid-1960s, two startling publications proved that virtually all cells freely exchange small neutral and charged molecules. Soon after, cell-cell communication by gap junction channels was reported. While gap junctions are the major means of cell-cell communication, in the early 1980s, evidence surfaced that some cells might also communicate via membrane pores. Questions were raised about the possible artifactual nature of the pores. However, early in this century, we learned that communication via membrane pores exists and plays a major role in medicine, as the structures involved, “tunneling nanotubes”, can rescue diseased cells by directly transferring healthy mitochondria into compromised cells and tissues. On the other hand, pathogens/cancer could also use these communication systems to amplify pathogenesis. Here, we describe the evolution of the discovery of these new communication systems and the potential therapeutic impact on several uncurable diseases.

Meaningful connections: Interrogating the role of physical fibroblast cell-cell communication in cancer.

As part of the connective tissue, activated fibroblasts play an important role in development and disease pathogenesis, while quiescent resident fibroblasts are responsible for sustaining tissue homeostasis. Fibroblastic activation is particularly evident in the tumor microenvironment where fibroblasts transition into tumor-supporting cancer-associated fibroblasts (CAFs), with some CAFs maintaining tumor-suppressive functions. While the tumor-supporting features of CAFs and their fibroblast-like precursors predominantly function through paracrine chemical communication (e.g., secretion of cytokine, chemokine, and more), the direct cell-cell communication that occurs between fibroblasts and other cells, and the effect that the remodeled CAF-generated interstitial extracellular matrix has in these types of cellular communications, remain poorly understood. Here, we explore the reported roles fibroblastic cell-cell communication play within the cancer stroma context and highlight insights we can gain from other disciplines.

Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing.

Simple SummaryBystander effects depend on direct cell-cell communication and/or paracrine signaling mediated by the release of soluble factors into the extracellular environment and may greatly influence therapy outcome. Although the limited data available suggest a role for intercellular gap junction channels, far less is known about the role of connexin hemichannels. Here, we investigated bystander effects induced by photodynamic therapy in syngeneic murine melanoma models in vivo. We determined that (i) photoactivation of a photosensitizer triggered calcium-dependent cell death pathways in both irradiated and bystander tumor cells; (ii) hemichannel activity and adenosine triphosphate release were key factors for the induction of bystander cell death; and (iii) bystander cell killing and antitumor response elicited by photodynamic therapy were greatly enhanced by combination treatment with S-nitrosoglutathione, which promoted hemichannel opening in these experimental conditions. Therefore, these findings in a preclinical model have important translational potential.In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.

Gap Junction Channelopathies and Calmodulinopathies. Do Disease-Causing Calmodulin Mutants Affect Direct Cell-Cell Communication?

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell–cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.

Hydrogen Peroxide-Induced Senescence Reduces the Wound Healing-Promoting Effects of Mesenchymal Stem Cell-Derived Exosomes Partially via miR-146a

Mesenchymal stem cells (MSCs) have beneficial effects on wound healing. MSCs function through direct cell-cell communication or indirectly through paracrine secretion of exosomes. Here, we found that MSC-derived exosomes had pro-wound healing effects via promotion of angiogenesis; however, this promoting effect was significantly reduced when senescence was induced in parental MSCs by hydrogen peroxide (H2O2). Further experiments showed that decreased miR-146a expression in exosomes derived from senescent MSCs (s-exo) contributed to these findings. In vitro, the pro-angiogenic effect of s-exo on tube formation in human umbilical vein endothelial cells was significantly reduced compared with that of exosomes derived from control MSCs (c-exo). In vivo, higher tube numbers and longer tube lengths were observed in the c-exo group compared with the s-exo group. Using microarray analysis, we found that miR-146a level in s-exo was lower than that in c-exo. Knockdown of miR-146a in c-exo decreased its capacity to promote angiogenesis, and overexpression of miR-146a in s-exo partially rescued its impaired pro-angiogenic capacity, thereby confirming that downregulation of miR-146a contributed to the reduced pro-wound healing capacity of s-exo. Our study is the first to demonstrate that cell senescence induced by H2O2 alters the pro-angiogenic ability of exosomes by modulating the expression of exosomal miRNAs, especially miR-146a, thus providing new insights into the correlation between parental cell state and exosome content and function.

Abstract 5997: Increased connexin 43 expression and gap junction communication correlates with invasion following reduced glucose metabolism in breast cancer cells

Abstract Dysregulation of gap junction intercellular communication (GJIC) is a common feature during cancer progression. GJIC is a means of direct cell-cell communication mediated by regulated membrane channels composed of connexin proteins. This communication is frequently lost between primary tumor cells but may be upregulated at secondary metastatic sites with stromal cells. Control of this process by cancer cells has been shown to facilitate aggressive qualities both in vitro and in vivo. During the process of metastasis, cells encounter numerous metabolic challenges that must be overcome, particularly during growth of primary tumors. In this study, we set out to evaluate if changes to cancer cell metabolism affect GJIC in breast cancer cells. To address this question, we generated a metabolic variant of the MDA-MB-231 cell line conditioned to grow in glucose-limiting conditions. These cells were grown in FBS supplemented RPMI with <0.130mM glucose compared to 2mM in control conditions for more than 4 weeks. Substantial cell death over this time revealed a small population of cells capable of surviving in glucose reduced conditions. Growth of these cells normalized following a period of quiescence and exhibited stable viability and proliferative capacity. Following STR validation, these cells were designated MDA-MB-231LG for their ability to grow in low glucose media. MDA-MB-231LG exhibited a larger and more rounded morphologic appearance with formation of strong cell-cell contacts as demonstrated by scanning electron microscopy in contrast to parental cells which showed a higher degree of membrane overlap. Further comparison of the two cell lines through western blot and immunofluorescence analysis of connexin 43, a major connexin expressed in breast tissue, revealed higher levels in MDA-MB-231LG and increased membrane localization. Using a double label dye transfer technique, the gap junction permeable fluorescent dye calcein showed increased movement from CM-DiI labeled donor cells into neighboring cells in MDA-MB-231LG indicating functional gap junction coupling while MDA-MB-231 had little to no dye movement. To evaluate phenotypic qualities, both cell lines were grown in Matrigel and MDA-MB-231LG displayed increased stellate morphology. Use of a Matrigel invasion chamber assay confirmed the increased invasive qualities with significantly more MDA-MB-231LG invading through the lower portion of the membrane compared to MDA-MB-231 parental cells. Our data demonstrate a clear upregulation of gap junction activity following metabolic adaptation to reduced glucose availability. It also suggests a possible connection between GJIC and invasive qualities in breast cancer cells and may represent an inducible phenotype that occurs in primary tumors when tumor growth limits blood vessel penetration and nutrient availability. Citation Format: Jennifer C. Jones, Amanda M. Miceli, Mary M. Chaudhry, Mallika A. Jai, Romel N. Pancho, Alan Lazzar, Bradley S. Taylor, Vishnupriya Bodempudi, Prarthana P. Jain, Sheeri Hanjra, Alexander E. Urban, Brian Zanotti, Ellen K. Kohlmeir, Thomas M. Bodenstine. Increased connexin 43 expression and gap junction communication correlates with invasion following reduced glucose metabolism in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5997.

Molecular Determinants of Gap Junction Channels Conductance

Gap junction (GJ) channels are formed by two hemichannels, each contributed by the cells taking part in this direct cell-cell communication conduit. These hydrophilic protein channels mediate the transfer of small cytoplasmic molecules between adjacent cells, such as ions and metabolites including second messengers. GJ channels formed by protein monomers, called connexins, are found in most cell types in vertebrates. These ubiquitous yet specific proteins form poorly selective channels with unique properties, and differ in their ability to mediate the transfer of specific molecules. In order to provide insight into molecular determinants of GJ channels conductance, we used molecular crystal structures of Connexin 26 (Cx26, also known as GJB2), PDB ID: 2ZW3, and Connexin 50 (Cx50, also known as GJA8), PDB ID: 6MHY. Molecular dynamics simulations were conducted with an external applied electric field to test intercellular communication via these channels evaluating conductance and selectivity. We compared pore radius and electrostatic profiles of both channels. Also, we compared the values of the conductance estimated by the observed permeation events with those reported in the literature. Our preliminary results suggest that electrostatic profile is a key determinant of ion selectivity and conductance. GJ channels formed by Cx26 show fewer diffusion events than those formed by Cx50, which corresponds with conductance values reported in the literature. Finally, we identified energy barriers for passage of ions through each channel and elucidated the effect of pore structure on energy barriers. This work was supported by the following grants: CONICYT PFCHA/Doctorado 21161628 (to C. Pareja-Barrueto),FONDECYT Regular 1170733 and ICM-MINECON P09-022-F (to D. Gonzalez-Nilo and J. C. Sáez), Anillo ATC71 and FONDECYT Regular 1191329 (to J. C. Sáez).

Biological Functions of Connexin43 Beyond Intercellular Communication.

Connexin43 (Cx43) is commonly associated with direct cell-cell communication through gap junctions (GJs). However, recent groundbreaking studies have challenged this dogma, implicating Cx43 in other biological processes, such as transcription, metabolism, autophagy, and ion channel trafficking. How Cx43 participates in these processes remains largely unknown, although its high turnover rate, capacity to bind to myriad proteins, and the discovery of truncated isoforms of Cx43, ascribe to this protein unanticipated roles in chief processes that require fine-tuned regulation. Accordingly, Cx43 can be regarded as a central integrative hub to which diverse cues converge to be processed in a concerted manner. In this review, we examine the noncanonical roles of Cx43 and discuss the implications of these functions in human diseases and future therapeutic strategies.

Mature Osteoblasts Provide a Protective Niche Against Multiple Myeloma Growth and Survival within the Tumor Microenvironment

Multiple myeloma (MM), a tumor of B-lymphocyte lineage cells, originates in the bone marrow (BM) and is highly influenced by the BM tumor microenvironment (TME). Among cells of the TME, osteoblasts are the most versatile regulators of many hematopoietic lineage cells through either direct cell-cell communication or secreted factors. Specifically relevant to MM, G-protein coupled receptor signaling in pre-osteoblasts is essential for the differentiation, maturation, and egress of B-cells (Panaroni et al., 2015). Despite these key roles, the contribution of osteoblasts to the initiation and progression of MM is not well understood. MM is characterized by osteolytic bone lesions partly due to decreased numbers of osteoblasts. Here, we hypothesize that osteoblasts provide niche support to maintain myeloma cells in a quiescent stage and that the loss of the osteoblastic niche leads to the progression of MM. As a proof of concept, we previously showed that increasing osteoblastogenesis by inhibiting Activin A led to inhibition of MM growth in an in vivo humanized myeloma model (Vallet et al., 2010).We generated mice in which mature osteoblasts could be postnatally deleted in an inducible and reversible manner. Diphtheria toxin receptor floxed mice were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter to generate OC-Cre/iDTR mice. Littermates heterozygous for DTR but lacking the OC-Cre expression were used as controls. The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/Kg DT beginning at 8-weeks of age. Osteocalcin immunohistochemistry of trabecular bone showed that the DT treated OC-Cre/iDTR mice were completely devoid of endosteal osteoblasts and young osteocytes. Consequently, serum levels of sclerostin were also significantly reduced in OC-Cre/iDTR mice compared to the controls.To study MM engraftment and progression, 3x106 5TGM1-Luciferase MM cells were inject into tibia of OC-Cre/iDTR and control mice followed by a weekly injection of DT for 8-weeks. Bioluminescence imaging (BLI) was used to assess tumor progression. Both the control and OC-Cre/iDTR mice started with similar BLI signal at 1-week. Interestingly, 4-weeks onwards only the OC-iDTR mice continued to express and increase the BLI signal indicating that the MM cells engrafted and continued to proliferate only in the OC-Cre/iDTR mice. This data suggests that under physiological conditions mature osteoblasts actively suppress MM engraftment and progression.To determine the direct effects of osteoblasts on MM cells, we established co-cultures of osteoblasts with MM cells. FACS sorting was used to isolate pre-osteoblasts from the long bones of Osterix-GFP+ (Osx+) mice, committed osteoblasts from Collagen 2.3-GFP+ (Col2.3+) mice, and mature osteoblasts from Osteocalcin-YFP+ (OCN+) mice. 5TGM1 MM cells were co-cultured along with 10,000 cells from each of the osteoblast populations. Although all three populations of osteoblasts significantly suppressed MM proliferation, the OCN+ mature osteoblasts suppressed MM proliferation the most.The mature osteoblastic niche may regulate MM cells via a) direct cell-to-cell contact, b) secreted factors, and/or c) other intermediary cells. To identify these possibilities, the BM TME was examined at 2-weeks following the 5TGM1 intratibia injection into the OC-Cre/iDTR and control mice. Quantitative protein antibody arrays analysis on the BM supernatant identified numerous key factors involved in cell-cell communication and immunomodulation in MM. These factors included BAFF-R, TACI, IL-33, IL-3, IL-21, and IL-17F. Gene expression analysis of the sorted 5TGM1 cells from the injected tibia indicated increased expression of genes involved in mitochondrial metabolism.Taken together, using in vivo and in vitro models, we show that mature osteoblasts offer specialized niches for MM cells where tumor cells are maintained in quiescence. The loss of the niche support allows the reactivation and progression of MM through the loss of cell-cell communication or through activation of immunomodulatory intermediary cells. Normalizing osteoblasts, such as through Activin A treatment, could provide novel avenues to reduce disease burden and long term tumor control. DisclosuresNo relevant conflicts of interest to declare.

Direct cell-cell communication with three-dimensional cell morphology on wrinkled microposts

Cell-cell communication plays a critical role in a myriad of processes, such as homeostasis, angiogenesis, and carcinogenesis, in multi-cellular organisms. Monolayer cell models have notably improved our understanding of cellular interactions. However, the cultured cells on the planar surfaces adopt a two-dimensional morphology, which poorly imitates cellular organization in vivo, providing physiologically-irrelevant cell responses. Non-planar surfaces comprising various patterns have demonstrated great abilities in directing cellular growth and producing different cell morphologies. In recent years, a few topographical substrates have provided valuable information about cell-cell signalling, however, none of these studies have reported a three-dimensional (3D) cell morphology. Here, we introduce a structurally tunable topographical platform that can maintain cell coupling while inducing a true 3D cell morphology. Optical imaging and fluorescence recovery after photobleaching are used to illustrate these capabilities. Our analyses suggest that the intercellular signalling on the present platform, which we propose is mainly through gap junctions, is comparable to that in natural tissue. Statement of SignificanceA better understanding of direct cellular communication can help treating neurological diseases and cancers, which may be caused by dysfunctional intercellular signaling. To investigate cell-cell contact, cells are conventionally plated onto planar surfaces, where they flatten and adopt a two-dimensional cell morphology. These unrealistic models are physiologically-irrelevant since cells exhibit a three-dimensional (3D) shape in the body. Therefore, porous scaffolds and topographical surfaces, capable of inducing various cell morphologies, have been introduced, in which the latter is more desirable for sample imaging and screening. However, the few non-planar substrates used to study cell coupling have not produced a 3D cell shape. Here, we present a tunable culture platform that can control direct cell-cell communication while maintaining true 3D cell morphologies.

Connexin 43 K63-polyubiquitylation on lysines 264 and 303 regulates gap junction internalization

Gap junctions (GJs) assembled from connexin (Cx) proteins allow direct cell-cell communication. While phosphorylation is known to regulate multiple GJ functions, much less is known about the role of ubiquitin in these processes. Using ubiquitylation-type-specific antibodies and Cx43 lysine-to-arginine mutants we show that ∼8% of a GJ, localized in central plaque domains, is K63-polyubiquitylated on K264 and K303. Levels and localization of ubiquitylation correlated well with: (1) the short turnover rate of Cxs and GJs; (2) removal of older channels from the plaque center; and (3) the fact that not all Cxs in an internalizing GJ channel need to be ubiquitylated. Connexins mutated at these two sites assembled significantly larger GJs, exhibited much longer protein half-lives and were internalization impaired. Interestingly, these ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitylation is triggered by phosphorylation. Phospho-specific anti-Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282 and 255, which are well-known regulatory PKC and MAPK sites. Together, these novel findings suggest that the internalizing portion of channels in a GJ is K63-polyubiquitylated, ubiquitylation is critical for GJ internalization and that phosphorylation induces Cx K63-polyubiquitylation.This article has an associated First Person interview with the first author of the paper.

Deregulation of the Notch pathway as a common road in viral carcinogenesis.

The Notch pathway is a conserved signaling pathway and a form of direct cell-cell communication related to many biological processes during development and adulthood. Deregulation of the Notch pathway is involved in many diseases, including cancer. Almost 20% of all cancer cases have an infectious etiology, with viruses responsible for at least 1.5 million new cancer cases per year. Seven groups of viruses have been classified as oncogenic: hepatitis B and C viruses (HBV and HCV respectively), Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV), human T lymphotropic virus (HTLV-1), human papillomavirus (HPV), and Merkel cell polyomavirus (MCPyV). These viruses share the ability to manipulate a variety of cell pathways that are critical in proliferation and differentiation, leading to malignant transformation. Viral proteins interact directly or indirectly with different members of the Notch pathway, altering their normal function. This review focuses exclusively on the direct interactions of viral oncoproteins with Notch elements, providing a deeper understanding of the dual behavior of the Notch pathway as activator or suppressor of neoplasia in virus-related cancers.

Aberrant Expression of Cx Isoforms in the Adult Caput Epididymis exposed to Estradiol Benzoate or Flutamide at the Weaning.

ABSTRACTConnexin (Cx) involves in the regulation of various physiological functions of tissue by forming a channel, a gap junction which allows direct cell-cell communication, between adjacent cells. The effect of a single subcutaneous treatment of estradiol benzoate (EB) or flutamide (Flu) at the weaning age on the expression of Cx isoforms in the adult caput epididymis was evaluated in this research. Using quantitative real-time PCR analysis, a low-dose of EB [0.015 μg/kg body weight (BW)] caused significant decreases of Cx30.3, Cx32, Cx40, Cx43, and Cx45 mRNA levels and no change of Cx26, Cx31, Cx31.1, Cx37 transcript levels. The treatment of a high-dose EB (1.5 μg/kg BW) resulted in reduced expression of Cx30.3, Cx31, Cx43, and Cx45 but increased expression of Cx37 and Cx40. Expression of all Cx isoforms examined, except Cx31, was significantly increased by the treatment of a low-dose Flu (500 μg/kg BW). However, the treatment of a high-dose Flu (5 mg/kg BW) led significant expressional suppression of Cx30.3, Cx31, Cx31.1, Cx32, Cx40, Cx43, and Cx45 but an increase of Cx37 transcript level. With the comparison of previous findings, the expression of Cx isoforms in the adult epididymis after the exposure to EB or Flu is likely differentially regulated in regional-specific and/or exposed postnatal age-specific manner.

Gap junctional intercellular communication in adipose-derived stromal/stem cells is cell density-dependent and positively impacts adipogenic differentiation.

Adipose-derived stromal/stem cells (ASCs) represent a widely used cell source with multi-lineage differentiation capacity in approaches for tissue engineering and regenerative medicine. Despite the multitude of literature on their differentiation capacity, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction-forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in human ASCs, and its significance for adipogenic differentiation of these cells. Cx43 expression in ASCs was demonstrated histologically and on the gene and protein expression level, and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of ASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α-glycyrrhetinic acid (AGA) significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process. Altogether, this study demonstrates the impact of direct cell-cell communication via gap junctions on the adipogenic differentiation process of ASCs, and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches.

Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action.Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies.