Muscarinic acetylcholine receptor-mediated phosphorylation of extracellular signal-regulated kinase in HSY salivary ductal cells involves distinct signaling pathways.

OBJECTIVES: Typical agonists of G protein-coupled receptors (GPCRs), including muscarinic acetylcholine receptors (mAChRs), activate both G-protein and ß-arrestin signaling systems, and are termed balanced agonists. In contrast, biased agonists selectively activate a single pathway, thereby offering therapeutic potential for the specific activation of that pathway. The mAChR agonists carbachol and pilocarpine are known to induce phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) via G-protein-dependent and -independent pathways, respectively. We investigated the involvement of ß-arrestin and its downstream mechanisms in the ERK1/2 phosphorylation induced by carbachol and pilocarpine in the human salivary ductal cell line, HSY cells. METHODS: HSY cells were stimulated with pilocarpine or carbachol, with or without various inhibitors. The cell lysates were analyzed by western blotting using the antibodies p44/p42MAPK and phosphor-p44/p42MAPK. RESULTS: Western blot analysis revealed that carbachol elicited greater stimulation of ERK1/2 phosphorylation compared to pilocarpine. ERK1/2 phosphorylation was inhibited by atropine and gefitinib, suggesting that mAChR activation induces transactivation of epidermal growth factor receptors (EGFR). Moreover, inhibition of carbachol-mediated ERK1/2 phosphorylation was achieved by GF-109203X (a PKC inhibitor), a ßARK1/GRK2 inhibitor, barbadin (a ß-arrestin inhibitor), pitstop 2 (a clathrin inhibitor), and dynole 34-2 (a dynamin inhibitor). In contrast, pilocarpine-mediated ERK1/2 phosphorylation was only inhibited by barbadin (a ß-arrestin inhibitor) and PP2 (a Src inhibitor). CONCLUSION: Carbachol activates both G-protein and ß-arrestin pathways, whereas pilocarpine exclusively activates the ß-arrestin pathway. Additionally, downstream of ß-arrestin, carbachol activates clathrin-dependent internalization, while pilocarpine activates Src.

Engineering of an electrically charged hydrogel implanted into a traumatic brain injury model for stepwise neuronal tissue reconstruction.

Neural regeneration is extremely difficult to achieve. In traumatic brain injuries, the loss of brain parenchyma volume hinders neural regeneration. In this study, neuronal tissue engineering was performed by using electrically charged hydrogels composed of cationic and anionic monomers in a 1:1 ratio (C1A1 hydrogel), which served as an effective scaffold for the attachment of neural stem cells (NSCs). In the 3D environment of porous C1A1 hydrogels engineered by the cryogelation technique, NSCs differentiated into neuroglial cells. The C1A1 porous hydrogel was implanted into brain defects in a mouse traumatic damage model. The VEGF-immersed C1A1 porous hydrogel promoted host-derived vascular network formation together with the infiltration of macrophages/microglia and astrocytes into the gel. Furthermore, the stepwise transplantation of GFP-labeled NSCs supported differentiation towards glial and neuronal cells. Therefore, this two-step method for neural regeneration may become a new approach for therapeutic brain tissue reconstruction after brain damage in the future.

Insertion of circularly permuted cyan fluorescent protein into the ligand-binding domain of inositol 1,4,5-trisphosphate receptor for enhanced FRET upon binding of fluorescent ligand.

Binding of fluorescent ligand (FL) to the cyan fluorescent protein (CFP)-coupled ligand-binding domain of the inositol 1,4,5-trisphosphate (IP3) receptor (CFP-LBP) produces fluorescence (Förster) resonance energy transfer (FRET). A competitive fluorescent ligand assay (CFLA), using the FRET signal from competition between FLs and IP3, can measure IP3 concentration. The FRET signal should be enhanced by attaching a FRET donor to an appropriate position. Herein, we inserted five different circularly permuted CFPs in the loop between the second and third α-helices to generate membrane-targeted fluorescent ligand-binding proteins (LBPs). Two such proteins, LBP-cpC157 and LBP-cpC173, localized at the plasma membrane, displayed FRET upon binding the high-affinity ligand fluorescent adenophostin A (F-ADA), and exhibited a decreased fluorescence emission ratio (480 nm / 535 nm) by 1.6- to 1.8-fold that of CFP-LBP. In addition, binding of a fluorescent low-affinity ligand (F-LL) also reduced the fluorescence ratio in a concentration-dependent manner, with EC50 values for LBP-cpC157 and LBP-cpC173 of 34.7 nM and 27.6 nM, respectively. These values are comparable to that with CFP-LBP (29.2 nM), indicating that insertion of cpC157 and cpC173 did not disrupt LBP structure and function. The effect of 100 nM F-LL on the decrease in fluorescence ratio was reversed upon addition of IP3, indicating binding competition between F-LL and IP3. We also constructed cytoplasmic fluorescent proteins cyLBP-cpC157 and cyLBP-cpC173, and bound them to DYK beads for imaging analyses. Application of F-ADA decreased the fluorescence ratio of the beads from the periphery to the center over 3 - 5 min. Application of F-LL also decreased the fluorescence ratio of cyLBP-cpC157 and cyLBP-cpC173 by 20-25%, and subsequent addition of IP3 recovered the fluorescence ratio in a concentration-dependent manner. The EC50 value and Hill coefficient obtained by curve fitting against the IP3-dependent recovery of fluorescence ratio can be used to estimate the IP3 concentration.

Spontaneous calcium responses of SF2 rat dental epithelial cells stably expressing the calcium sensor G-GECO.

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.

Rapid reprogramming of tumour cells into cancer stem cells on double-network hydrogels.

Cancer recurrence can arise owing to rare circulating cancer stem cells (CSCs) that are resistant to chemotherapies and radiotherapies. Here, we show that a double-network hydrogel can rapidly reprogramme differentiated cancer cells into CSCs. Spheroids expressing elevated levels of the stemness genes Sox2, Oct3/4 and Nanog formed within 24 h of seeding the gel with cells from any of six human cancer cell lines or with brain cancer cells resected from patients with glioblastoma. Human brain cancer cells cultured on the double-network hydrogel and intracranially injected in immunodeficient mice led to higher tumorigenicity than brain cancer cells cultured on single-network gels. We also show that the double-network gel induced the phosphorylation of tyrosine kinases, that gel-induced CSCs from primary brain cancer cells were eradicated by an inhibitor of the platelet-derived growth factor receptor, and that calcium channel receptors and the protein osteopontin were essential for the regulation of gel-mediated induction of stemness in brain cancer cells.

Synthetic poly(2-acrylamido-2-methylpropanesulfonic acid) gel induces chondrogenic differentiation of ATDC5 cells via a novel protein reservoir function.

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.

Integrin α4 mediates ATDC5 cell adhesion to negatively charged synthetic polymer hydrogel leading to chondrogenic differentiation.

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.

Exosomes containing ErbB2/CRK induce vascular growth in premetastatic niches and promote metastasis of bladder cancer.

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.

Micro patterning of hydroxyapatite by soft lithography on hydrogels for selective osteoconduction.

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.

Synthetic PAMPS gel activates BMP/Smad signaling pathway in ATDC5 cells, which plays a significant role in the gel-induced chondrogenic differentiation.

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-ß/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-ß/BMP signaling pathway significantly inhibited the chondrogenic differentiation induced by the PAMPS gel. The present study demonstrated that synthetic PAMPS gel activates the TGF-ß/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.