Osmotic gradients induce stable dome morphogenesis on extracellular matrix.

One of the fundamental processes in morphogenesis is dome formation, but many of the mechanisms involved are unexplored. Previous in vitro studies showed that an osmotic gradient is the driving factor of dome formation. However, these investigations were performed without extracellular matrix (ECM), which provides structural support to morphogenesis. With the use of ECM, we observed that basal hypertonic stress induced stable domes in vitro that have not been seen in previous studies. These domes developed as a result of ECM swelling via aquaporin water transport activity. Based on computer simulation, uneven swelling, with a positive feedback between cell stretching and enhanced water transport, was a cause of dome formation. These results indicate that osmotic gradients induce dome morphogenesis via both enhanced water transport activity and subsequent ECM swelling.

Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts.

Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner.

The intercellular expression of type-XVII collagen, laminin-332, and integrin-ß1 promote contact following during the collective invasion of a cancer cell population.

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-ß1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-ß1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-ß1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-ß1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.

Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells.

Glycans, including glycosphingolipids, are broadly expressed in plasma membranes and play important roles in cell-cell interactions. Recently, it has been revealed that glycans participate in the regulation of malignant phenotypes of cancer cells, e.g. growth and invasion. However, their roles in irradiation-tolerant cancer cells have not yet been elucidated. In this study, we show that specific glycosphingolipids are highly expressed in invasive, irradiation-tolerant lung cancer cells. Particularly, the glycosphingolipid GM2 contributes to the development of an invasive phenotype in these lung cancer cells. Our results suggest that glycosphingolipids, including GM2, are implicated in the regulation of invasiveness in irradiation-tolerant lung cancer cells and may therefore serve as potential therapeutic targets for lung cancers following radiotherapy.Key words: glycosphingolipids, GM2, invasion, lung cancer cells, radiotherapy.

Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization.

Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. Here, we studied the role of the actin-binding protein Girdin, a specific regulator of collective migration of neuroblasts in the brain, in collective cancer cell migration. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast-led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to ß-catenin that plays important roles in the Wnt signaling pathway and in E-cadherin-mediated cell-cell adhesion. Girdin-depleted cells displayed scattering and impaired E-cadherin-specific cell-cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the ß-catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell-cell adhesion in the collective behavior of cells. Finally, we showed the correlation of the expression of Girdin with that of the components of the E-cadherin complex and the differentiation of human skin cancer. Collectively, our results suggest that Girdin is an important modulator of the collective behavior of cancer cells.

Mathematical model of collective cell migrations based on cell polarity.

Individual cells migrate toward the direction of the cell polarity generated by interior or exterior factors. Under situations without guides such as chemoattractants, they migrate randomly. On the other hand, it has been observed that cell groups lead to systematic collective cell migrations. For example, Dictyostelium discoideum and Madin-Darby canine kidney (epithelial) cells exhibit typical collective cell migration patterns such as uniformly directional migration and rotational migration. In particular, it has been suggested from experimental investigations that rotational migrations are intimately related to morphogenesis of organs and tissues in several species. Thus, it is conjectured that collective cell migrations are controlled by universal mechanisms of cells. In this paper, we review actual experimental data related to collective cell migrations on dishes and show that our self-propelled particle model based on the cell polarity can accurately represent actual migration behaviors. Furthermore, we show that collective cell migration modes observed in our model are robust.

Role of ATF5 in the invasive potential of diverse human cancer cell lines.

Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element-binding protein family. Our research group recently revealed that ATF5 expression increases the invasiveness of human lung carcinoma cells. However, the effects of ATF5 on the invasive potential of other cancer cells lines remain unclear. Therefore, in this study, we investigated the role of ATF5 in the invasive activity of diverse human cancer cell lines. Invasiveness was assessed using Matrigel invasion assays. ATF5 knockdown resulted in decreased invasiveness in seven of eight cancer cell lines tested. These results suggest that ATF5 promotes invasiveness in several cancer cell lines. Furthermore, the roles of ATF5 in the invasiveness were evaluated in three-dimensional (3D) culture conditions. In 3D collagen gel, HT-1080 and MDA-MB-231 cells exhibited high invasiveness, with spindle morphology and high invasion speed. In both cell lines, knockdown of ATF5 resulted in rounded morphology and decreased invasion speed. Next, we showed that ATF5 induced integrin-α2 and integrin-ß1 expression and that the depletion of integrin-α2 or integrin-ß1 resulted in round morphology and decreased invasion speed. Our results suggest that ATF5 promotes invasion by inducing the expression of integrin-α2 and integrin-ß1 in several human cancer cell lines.

Filamin B Enhances the Invasiveness of Cancer Cells into 3D Collagen Matrices.

Numerous types of cancer cells migrate into extracellular tissues. This phenomenon is termed invasion, and is associated with poor prognosis in cancer patients. In this study, we demonstrated that filamin B (FLNb), an actin-binding protein, is highly expressed in cancer cell lines that exhibit high invasiveness, with a spindle morphology, into 3D collagen matrices. In addition, we determined that knockdown of FLNb in invasive cancer cells converts cell morphology from spindle-shaped, which is associated with high invasiveness, to round-shaped with low invasiveness. Furthermore, di-phosphorylation of myosin regulatory light chain (MRLC) and phosphorylation of focal adhesion kinase (FAK) are inhibited in FLNb-knockdown cancer cells. These results suggest that FLNb enhances invasion of cancer cells through phosphorylation of MRLC and FAK. Therefore, FLNb may be a new therapeutic target for invasive cancers.

Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85.

The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force.

Modulation of extracellular conditions prevents the multilayering of the simple epithelium.

Simple epitheliums in normal glandular systems are regulated not to stratify even though the constituent cells proliferate and will rise from the epithelium. Since epithelial cells have the potential to establish cell-cell adhesions, the avoidance of stratification must be related to the intracellular signal cascades and the extracellular conditions. The contributions of the former are becoming clarified, but the influence of the latter is poorly understood. In the present study, we examined whether the frequency of cell-on-cell adhesion, which mimics the early stage of multilayering, is dependent on the type of the extracellular scaffold protein. Wild-type epithelial cells were cultured on E-cadherin-Fc (a cell-cell adhesion protein) or collagen (an extracellular matrix protein), and then, green fluorescent protein (GFP)-positive cells were seeded onto these wild-type cells. We observed that the cell-on-cell adhesion (adhesion of the GFP-positive cell to the wild-type cells) was more frequent in the E-cadherin-Fc treatment than the collagen treatment. The cell-on-cell adhesions that were observed in the E-cadherin treatment were transient and decreased in frequency to that of the collagen treatment after the 12 h of cell culture. We observed the disappearance of E-cadherin-Fc but not collagen during cell culture. These results suggest that transient multilayering in simple epithelium is possible, depending on the types of extracellular scaffold protein, and they imply that cells can modify the extracellular conditions to meet normal cellular conditions.

Substrate stiffness regulates temporary NF-κB activation via actomyosin contractions.

Physical properties of the extracellular matrix (ECM) can control cellular phenotypes via mechanotransduction, which is the process of translation of mechanical stresses into biochemical signals. While current research is clarifying the relationship between mechanotransduction and cytoskeleton or adhesion complexes, the contribution of transcription factors to mechanotransduction is not well understood. The results of this study revealed that the transcription factor NF-κB, a major regulator for immunoreaction and cancer progression, is responsive to substrate stiffness. NF-κB activation was temporarily induced in H1299 lung adenocarcinoma cells grown on a stiff substrate but not in cells grown on a soft substrate. Although the activation of NF-κB was independent of the activity of integrin ß1, an ECM-binding protein, the activation was dependent on actomyosin contractions induced by phosphorylation of myosin regulatory light chain (MRLC). Additionally, the inhibition of MRLC phosphorylation by Rho kinase inhibitor Y27632 reduced the activity of NF-κB. We also observed substrate-specific morphology of the cells, with cells grown on the soft substrate appearing more rounded and cells grown on the stiff substrate appearing more spread out. Inhibiting NF-κB activation caused a reversal of these morphologies on both substrates. These results suggest that substrate stiffness regulates NF-κB activity via actomyosin contractions, resulting in morphological changes.

Interruption of glycosphingolipid synthesis enhances osteoarthritis development in mice.

OBJECTIVE: Glycosphingolipids (GSLs) are ubiquitous membrane components that modulate transmembrane signaling and mediate cell-to-cell and cell-to-matrix interactions. GSL expression is decreased in the articular cartilage of humans with osteoarthritis (OA). This study was undertaken to determine the functional role of GSLs in cartilage metabolism related to OA pathogenesis in mice. METHODS: We generated mice with knockout of the chondrocyte-specific Ugcg gene, which encodes an initial enzyme of major GSL synthesis, using the Cre/loxP system (Col2-Ugcg(-/-) mice). In vivo OA and in vitro cartilage degradation models were used to evaluate the effect of GSLs on the cartilage degradation process. RESULTS: Although Col2-Ugcg(-/-) mice developed and grew normally, OA changes in these mice were dramatically enhanced with aging, through the overexpression of matrix metalloproteinase 13 and chondrocyte apoptosis, compared to their wild-type (WT) littermates. Col2-Ugcg(-/-) mice showed more severe instability-induced pathologic OA in vivo and interleukin-1α (IL-1α)-induced cartilage degradation in vitro. IL-1α stimulation of chondrocytes from WT mice significantly increased Ugcg messenger RNA expression and up-regulated GSL metabolism. CONCLUSION: Our results indicate that GSL deficiency in mouse chondrocytes enhances the development of OA. However, this deficiency does not affect the development and organization of cartilage tissue in mice at a young age. These findings indicate that GSLs maintain cartilage molecular metabolism and prevent disease progression, although GSLs are not essential for chondrogenesis of progenitor and stem cells and cartilage development in young mice. GSL metabolism in the cartilage is a potential target for developing a novel treatment for OA.

Stiffness-Modulation of Collagen Gels by Genipin-Crosslinking for Cell Culture.

The stiffness of extracellular matrices (ECMs) is critical for cellular functions. Therefore, modulating the stiffness of ECMs in vitro is necessary to investigate the role of stiffness in cellular phenomena. Collagen gels are widely used for cell culture matrices in vitro. However, modulation of the stiffness in collagen gels for cell culture is challenging owing to the limited knowledge of the method to increase the stiffness while maintaining low cytotoxicity. Here, we established a novel method to modulate collagen gel stiffness from 0.0292 to 12.5 kPa with low cytotoxicity. We prepared collagens with genipin, a low-cytotoxic crosslinker of amines, at different concentrations and successfully modulated the stiffness of the gels. In addition, on 10 mM genipin-mixed collagen gels (approximately 12.5 kPa), H1299 human lung cancer cells showed spreading morphology and nuclear localization of yes-associated protein (YAP), typical phenomena of cells on stiff ECMs. Mouse mesenchymal stromal cells on 10 mM genipin-mixed collagen gels differentiated to vascular smooth muscle cells. On the other hand, the cells on 0 mM genipin-mixed collagen gels (approximately 0.0292 kPa) differentiated to visceral smooth muscle cells. Our new method provides a novel way to prepare stiffness-modulated collagen gels with low cytotoxicity in cell culture.

Substrate stiffness induces nuclear localization of myosin regulatory light chain to suppress apoptosis.

Stiffness of the extracellular matrix regulates various biological responses, but the response mechanisms are poorly understood. Here, we found that the nuclear diphosphorylated myosin regulatory light chain (2P-MRLC) is a critical mechanomediator that suppresses apoptosis in response to substrate stiffness. Stiff substrates promoted the nuclear localization of 2P-MRLC. Zipper-interacting protein kinase [ZIPK; also known as death-associated protein kinase 3 (DAPK3)], a kinase for MRLC, was localized in the nucleus in response to stiff substrates and promoted the nuclear localization of 2P-MRLC. Moreover, actin fiber formation induced by substrate stiffness promoted the nuclear localization of 2P-MRLC via ZIPK. 2P-MRLC in response to substrate stiffness suppressed the expression of MAF bZIP transcription factor B (MafB) and repressed apoptosis. These findings reveal a newly identified role of MRLC in mechanotransduction.

The EGF/EGFR axis and its downstream signaling pathways regulate the motility and proliferation of cultured oral keratinocytes.

We previously reported that the cell and colony motion of oral keratinocytes are correlated with proliferative capacity, and speculated that this may be a specific index for monitoring cell quality. However, how cell motility and proliferation are regulated by signaling pathways remains unelucidated. Here, we found that the regulation of cell motility and proliferative capacity of oral keratinocytes can be attributed to the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis. The EGFR downstream cascade involving the Src/PI3K/Akt/mTOR signaling pathway showed a major effect on cell motility and proliferative capacity in oral keratinocytes. Furthermore, both EGFR and Src attenuated E-cadherin expression. Taken together, these findings provide a potential basis for future quality control of cells for therapeutic use.

Matrix Stiffness Contributes to Cancer Progression by Regulating Transcription Factors.

Matrix stiffness is critical for the progression of various types of cancers. In solid cancers such as mammary and pancreatic cancers, tumors often contain abnormally stiff tissues, mainly caused by stiff extracellular matrices due to accumulation, contraction, and crosslinking. Stiff extracellular matrices trigger mechanotransduction, the conversion of mechanical cues such as stiffness of the matrix to biochemical signaling in the cells, and as a result determine the cellular phenotypes of cancer and stromal cells in tumors. Transcription factors are key molecules for these processes, as they respond to matrix stiffness and are crucial for cellular behaviors. The Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) is one of the most studied transcription factors that is regulated by matrix stiffness. The YAP/TAZ are activated by a stiff matrix and promotes malignant phenotypes in cancer and stromal cells, including cancer-associated fibroblasts. In addition, other transcription factors such as ß-catenin and nuclear factor kappa B (NF-κB) also play key roles in mechanotransduction in cancer tissues. In this review, the mechanisms of stiffening cancer tissues are introduced, and the transcription factors regulated by matrix stiffness in cancer and stromal cells and their roles in cancer progression are shown.

Improvement of the cell viability of hepatocytes cultured in three-dimensional collagen gels using pump-free perfusion driven by water level difference.

Cell-containing collagen gels are one of the materials employed in tissue engineering and drug testing. A collagen gel is a useful three-dimensional (3D) scaffold that improves various cell functions compared to traditional two-dimensional plastic substrates. However, owing to poor nutrient availability, cells are not viable in thick collagen gels. Perfusion is an effective method for supplying nutrients to the gel. In this study, we maintained hepatocytes embedded in a 3D collagen gel using a simple pump-free perfusion cell culture system with ordinary cell culture products. Flow was generated by the difference in water level in the culture medium. Hepatocytes were found to be viable in a collagen gel of thickness 3.26 (± 0.16 S.E.)-mm for 3 days. In addition, hepatocytes had improved proliferation and gene expression related to liver function in a 3D collagen gel compared to a 2D culture dish. These findings indicate that our perfusion method is useful for investigating the cellular functions of 3D hydrogels.

The interferon-ß/STAT1 axis drives the collective invasion of skin squamous cell carcinoma with sealed intercellular spaces.

The process by which cancer cells invade as a cell cluster, known as collective invasion, is associated with metastasis and worse prognosis of cancer patients; therefore, inhibition of collective invasion is considered to improve cancer treatment. However, the cellular characteristics responsible for collective invasion remain largely unknown. Here, we successfully established subclones with various invasive potentials derived from human skin squamous carcinoma cells. The cell cluster of the highly invasive subclone had a hermetically sealed and narrow intercellular space. Interferon-ß was localized to the sealed intercellular spaces, leading to collective invasion via the activation of signal transducer and activator of transcription 1 (STAT1). On the other hand, interferon-ß was not localized to non-sealed and wide intercellular spaces of the cell cluster of low-invasive subclone with deficient STAT1 activity. In the mixed cell cluster of high- and low-invasive subclones, the high-invasive sub-clonal cells were located at the invasive front of the invasive protrusion, leading to collective invasion by the low-invasive sub-clonal cells. Tissue microarray analysis of human skin squamous cell carcinoma (SCC) also showed enrichment of STAT1 in the invasive front of SCCs. These findings indicate that the intercellular structure controls the potential for collective invasion via STAT1 regulation in SCC.

Mammaglobin 1 mediates progression of trastuzumab-resistant breast cancer cells through regulation of cyclins and NF-κB.

Overexpression of human epidermal growth factor receptor 2 (HER2) in various cancers is correlated with poor patient survival. Trastuzumab, a recombinant humanized monoclonal antibody against HER2, has been considered to be a first-line therapy for HER2-positive breast cancer patients, but its usefulness is limited by the development of resistance. In this study, we established resistant cells by long-term treatment with trastuzumab. These cells showed higher proliferation, invasion, and migration abilities than the wild-type cells. Mammaglobin 1 (MGB1), cyclin D1, E1, A2, and phosphorylated NF-κB (p-p65) were upregulated in resistant cells. These proteins regulate cell proliferation, migration, and invasion of resistant cells. Depletion of MGB1 decreased cyclin and p-p65 expression. Cyclin D1 and A2, but not E1 expression, were affected by p-p65 downregulation. In summary, our results indicate that MGB1 expression is increased in breast cancer cells that have gained resistance to trastuzumab, and suggest that MGB1 promotes aggressiveness through cyclin and NF-κB regulation.