Search

Genetically-encoded calcium indicators such as G-GECO are useful for studying Ca2+ responses during long-term processes. In this study, we employed a lentiviral vector and established a rat dental epithelial cell line that stably expressed G-GECO (SF2-G-GECO). Ca2+ imaging analysis under cell culture conditions revealed that SF2-G-GECO cells exhibited spontaneous Ca2+ responses, which could be classified into the following three major patterns depending on the cell density: localized Ca2+ responses at cell protrusions at a low density, a cell-wide spread of Ca2+ responses at a medium density, and Ca2+ responses in clusters of 3-20 cells at a high density. The P2Y receptor inhibitor suramin (10 μM), the ATP-degrading enzyme apyrase (5 units/mL), and the fibroblast growth factor (FGF) receptor inhibitor FIIN-2 (1 μM) decreased the frequency of spontaneous Ca2+ responses. These results indicate that ATP and FGF are involved in the spontaneous Ca2+ responses. SF2 cells differentiate into ameloblasts via interactions with mesenchymal cells. Therefore, SF2-G-GECO cells are expected to be a useful tool for studying the functions of Ca2+ responses in regulating gene expression during tooth development.

We previously demonstrated that a synthetic negatively charged poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel induced chondrogenic differentiation of ATDC5 cells. In this study, we clarified the underlying molecular mechanism, in particular, focusing on the events that occurred at the interface between the gel and the cells. Gene expression profiling revealed that the expression of extracellular components was enhanced in the ATDC5 cells that were cultured on the PAMPS gel, suggesting that extracellular proteins secreted from the ATDC5 cells might be adsorbed in the PAMPS gel, thereby contributing to the induction of chondrogenic differentiation. Therefore, we created "Treated-PAMPS gel," which adsorbed various proteins secreted from the cultured ATDC5 cells during 7 days. Proteomic analysis identified 27 proteins, including extracellular matrix proteins such as Types I, III, and V collagens and thrombospondin (THBS) in the Treated-PAMPS gel. The Treated-PAMPS gel preferentially induced expression of chondrogenic markers, namely, aggrecan and Type II collagen, in the ATDC5 cells compared with the untreated PAMPS gel. Addition of recombinant THBS1 to the ATDC5 cells significantly enhanced the PAMPS-induced chondrogenic differentiation, whereas knockdown of THBS1 completely abolished this response. In conclusion, we demonstrated that the PAMPS gel has the potential to induce chondrogenic differentiation through novel reservoir functions, and the adsorbed THBS plays a significant role in the induction.

Locally advanced and metastatic invasive bladder cancer (BC) has a poor prognosis, and no advanced therapies beyond cisplatin‐based combination chemotherapy have been developed. Therefore, it is an urgent issue to elucidate the underlying mechanisms of tumor progression and metastasis of invasive BC for the development of new therapeutic strategies. Here, we clarified a novel role of exosomes containing ErbB2 and CRK in a formation of premetastatic niches and subsequent metastases. CRK adaptors were overexpressed in invasive UM‐UC‐3 BC cells. In an orthotopic xenograft model, metastases to lung, liver, and bone of UM‐UC‐3 cells were completely abolished by CRK elimination. Mass spectrometry analysis identified that ErbB2 was contained in UM‐UC‐3‐derived exosomes in a CRK‐dependent manner; the exosomes significantly increased proliferation and invasion properties of low‐grade 5637 BC cells and HUVECs through FAK and PI3K/AKT signaling pathways. In athymic mice educated with UM‐UC‐3‐derived exosomes, i.v. implanted UM‐UC‐3 cells were trapped with surrounding PKH67‐labeled exosomes in lung and led to development of lung metastasis with disordered vascular proliferation. In contrast, exosomes derived from CRK‐depleted BC cells failed to induce these malignant features. Taken together, we showed that CRK adaptors elevated the expression of ErbB2/3 in BC cells, and these tyrosine kinase/adaptor units were transferred from host BC cells to metastatic recipient cells by exosomes, leading to vascular leakiness and proliferation and contributing to the formation of distant metastasis. Thus, CRK intervention with ErbB2/3 blockade might be a potent therapeutic strategy for patients with ErbB2 overexpressing advanced and metastatic BC.

Negatively charged synthetic hydrogels have been known to facilitate various cellular responses including cell adhesion, proliferation, and differentiation; however, the molecular mechanism of hydrogel-dependent control of cell behavior remains unclear. Recently, we reported that negatively charged poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel induces chondrogenic differentiation of ATDC5 cells via novel protein reservoir function. In this study, we identified the cell adhesion molecules binding to PAMPS gels that act as mechanoreceptors. First, we performed a pull-down assay by particle gels using cell membrane proteins of ATDC5, and found that multiple membrane proteins bound to the PAMPS gel, whereas the uncharged poly(N,N'-dimethylacrylamide) gel as control did not bind to any membrane proteins. Western blot analysis indicated differential binding of integrin (ITG) isoforms to the PAMPS gel, in which the α4 isoform, but not α5 and αv, efficiently bound to the PAMPS gel. ITG α4 knockdown decreased cell spreading of ATDC5 on PAMPS gels, whereas the enhanced expression increased the behavior. Furthermore, ITG α4 depletion suppressed PAMPS gel-induced expression of bone morphogenic protein (BMP) 4 contributing to chondrogenic differentiation, in concordance with the reduction of ERK activation. These results demonstrated that membrane protein binding to PAMPS gels occurred in a charge-dependent manner, and that ITG α4 plays a crucial role in cell spreading on PAMPS gels and acts as a mechanoreceptor triggering cellular signaling leads to chondrogenic differentiation.

Mechanically robust hydrogels are promising biomaterials as artificial supportive tissue. These applications require selective and robust bonding of the hydrogels to living tissue. Recently, we achieved strong in vivo bone bonding of a tough double network (DN) hydrogel, a potential material for use as artificial cartilage and tendon, by hybridizing osteoconductive hydroxyapatite (HAp) in the gel surface layer. In this work, we report micro patterning of HAp at the surface of the DN hydrogel for selective osteoconduction. Utilizing the dissolution of HAp in an acidic environment, the soft lithography technique using an acid gel stamp was adopted to form a high-resolution HAp pattern on the gel. The HAp-patterned gel showed well-regulated migration and adhesion of cells in vitro. Moreover, the HAp-patterned gel showed selective and robust bonding to the rabbit bone tissue in vivo. This HAp soft lithography technique allows for simple and quick preparation of tailor-made osteoconductive hydrogels and can be applied to other hydrogels for selective bone bonding. STATEMENT OF SIGNIFICANCE: Hydrogels, preserving large amount of water, have been studied for next-generation artificial soft tissues. However, fixation of hydrogels to living tissue was unsolved issue for clinical application. Recently, we achieved robust bonding of a tough double network gel to bone in vivo by coating of osteoconductive hydroxyapatite in the gel surface layer. For further progress for practical use, we report the micro patterning of HAp at the surface of the DN hydrogel by using soft lithography technique, to perform selective bonding to only objective area without unnecessary coalescence. The HAp lithography technique is simple, quick and non-toxic method to prepare tailor-made osteoconductive hydrogels and has universality of species of hydrogels.

The purposes of this study were to identify signaling pathways that were specifically activated in ATDC5 cells cultured on poly (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel in insulin-free maintenance medium and to evaluate the significance of the determined signaling pathways in the chondrogenic differentiation induced by this gel. In this study, ATDC5 cells cultured on PAMPS gel using the maintenance medium without insulin (PAMPS Culture) were compared with cells cultured on polystyrene using the differentiation medium containing insulin (PS-I Culture). The microarray analysis, Western blot analysis, and real-time PCR analysis demonstrated that the TGF-β/BMP signaling pathway was significantly enhanced at Days 1, 2, and 3 in the PAMPS Culture when compared with the PS-I Culture. Inhibition of the BMP type-I receptor reduced the phosphorylation level of Smad1/5 and expression of type-2 collagen and aggrecan mRNA in the cells accompanied by a reduction in cell aggregation at Day 13 in the PAMPS Culture. The inhibition of the TGF-β/BMP signaling pathway significantly inhibited the chondrogenic differentiation induced by the PAMPS gel. The present study demonstrated that synthetic PAMPS gel activates the TGF-β/BMP/Smad signaling pathway in the ATDC5 cells in the absence of insulin, and that this activation plays a significant role in the chondrogenic differentiation induced by PAMPS gel. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 734-746, 2016.

Smooth muscle caldesmon (h-CaD) is an actin- and myosin-binding protein that reversibly inhibits the actomyosin ATPase activity in vitro. To test the function of h-CaD in vivo, we eliminated its expression in mice. The h-CaD-null animals appeared normal and fertile, although the litter size was smaller. Tissues from the homozygotes lacked h-CaD and exhibited up-regulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca2+-sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different. Both intact and permeabilized arterial smooth muscle tissues from the knockout animals relaxed more slowly than those of the wild-type. Since this difference occurred after myosin dephosphorylation was complete, the kinetic effect most likely resulted from slower detachment of unphosphorylated cross-bridges. Detailed analyses revealed that the apparently slower relaxation of h-CaD-null smooth muscle was due to an increase in the amplitude of a slower component of the biphasic tension decay. While the identity of this slower process has not been unequivocally determined, we propose it reflects a thin-filament state that elicits fewer re-attached cross-bridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.

Muscarinic acetylcholine receptors (mAChRs) are major regulators of gut epithelial functions. However, the precise subtype composition has not been clarified.We characterized the pharmacological profile of mAChRs on mouse colonic crypts, employing [(3)H]-N-methyl scopolamine chloride as a radioligand and several subtype-selective chemicals, and the functional aspect by measuring short-circuit current (I sc) in Ussing chambers and by evaluating MAP kinase phosphorylation in mouse colonic mucosal sheets.The mAChRs were detected on the crypts (K d = 163.2 ± 32.3 pM, B max = 47.3 ± 2.6 fmol/mg of total cell protein). Muscarinic toxin 7 (MT-7, M1 subtype selective) gave a displacement curve with high affinity, but there was a part insensitive to MT-7 (18.8 ± 0.4 % of the total specific binding). The MT-7-insensitive component was displaced completely by darifenacin (M3 selective) with high affinity. ACh induced an increase in I sc, which was significantly enhanced by MT-7 but was completely inhibited by darifenacin or atropine. Colitis induction resulted in a significant decrease in the density of mAChRs, which occurred mainly in the MT-7-sensitive component (M1 subtype). Immunological experiments exhibited a reduction of M1 but not of M3 signal after colitis induction. Muscarinic stimulation induced an increase in MAP kinase phosphorylation, which was completely suppressed by MT-7 and was attenuated by inflammation, in mouse colonic epithelium.These results suggest that mAChRs in mouse colonic epithelial cells consist of two subtypes, M1 (80 %) and M3 (20 %). The major M1 subtype was likely to regulate epithelial chloride secretion negatively and was susceptible to inflammation and may be relevant to inflammatory gut dysfunction.

We examined the change of protein tyrosine kinases (PTKs) expression levels in colonic epithelial cells isolated from mice in which colitis was induced by oxazolone administration, using the monoclonal antibody YK34, which cross-reacts with a wide variety of PTKs. We identified focal adhesion kinase (FAK) and found the expression level increased due to the induction of colitis. Furthermore, we found that there was a positive correlation between FAK expression and the severity of colitis. Also, FAK expression localized in the colonic epithelium but not in the lamina propria, implying FAK functions in epithelial cells during colitis formation and/or wound repairing.

The human polyomavirus JC virus (JCV) is the causative agent of a fatal demyelinating disease, progressive multifocal leukoencephalopathy, and encodes six major proteins, including agnoprotein. Agnoprotein colocalizes with microtubules in JCV-infected cells, but its function is not fully understood. We have now identified fasciculation and elongation protein zeta 1 (FEZ1) as a protein that interacted with JCV agnoprotein in a yeast two-hybrid screen of a human brain cDNA library. An in vitro binding assay showed that agnoprotein interacted directly with FEZ1 and microtubules. A microtubule cosedimentation assay revealed that FEZ1 also associates with microtubules and that agnoprotein induces the dissociation of FEZ1 from microtubules. Agnoprotein inhibited the promotion by FEZ1 of neurite outgrowth in PC12 cells. Conversely, overexpression of FEZ1 suppressed JCV protein expression and intracellular trafficking in JCV-infected cells. These results suggest that FEZ1 promotes neurite extension through its interaction with microtubules, and that agnoprotein facilitates JCV propagation by inducing the dissociation of FEZ1 from microtubules.