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.

Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury.

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction.

Disruption of epithelial barrier function was identified as one of the pathologic mechanisms in inflammatory bowel diseases (IBD). Epithelial barrier consists of various intercellular junctions, in which the tight junction (TJ) is an important component. However, the regulatory mechanism of tight junction is still not clear. Here we examined the role of focal adhesion kinase (FAK) in the epithelial barrier function on Caco-2 monolayers using a specific FAK inhibitor, PF-573, 228 (PF-228). We found that the decrease of transepithelial resistance and the increase of paracellular permeability were accompanied with the inhibition of autophosphorylation of FAK by PF-228 treatment. In addition, PF-228 inhibited the FAK phosphorylation at Y576/577 on activation loop by Src, suggesting Src-dependent regulation of FAK in Caco-2 monolayers. In an ethanol-induced barrier injury model, PF-228 treatment also inhibited the recovery of transepithelial resistance as well as these phosphorylations of FAK. In a sucrose gradient ultracentrifugation, FAK co-localized with claudin-1, an element of the TJ complex, and they co-migrate after ethanol-induced barrier injury. Immunofluorescence imaging analysis revealed that PF-228 inhibited the FAK redistribution to the cell border and reassembly of TJ proteins in the recovery after ethanol-induced barrier injury. Finally, knockdown of FAK by siRNA resulted in the decrease of transepithelial resistance. These findings reveal that activation of FAK is necessary for maintaining and repairing epithelial barrier in Caco-2 cell monolayer via regulating TJ redistribution.

Ablation of smooth muscle caldesmon affects the relaxation kinetics of arterial muscle.

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 upregulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca(2+) 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 crossbridges. 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 crossbridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

M1 is a major subtype of muscarinic acetylcholine receptors on mouse colonic epithelial cells.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) are major regulators of gut epithelial functions. However, the precise subtype composition has not been clarified. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Role of JC virus agnoprotein in virion formation.

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.

Oxazolone-induced over-expression of focal adhesion kinase in colonic epithelial cells of colitis mouse model.

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.

Nitric oxide-induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI-17 and MYPT1.

Nitric oxide (NO) from endothelium is a major mediator of vasodilatation through cGMP/PKG signals that lead to a decrease in Ca(2+) concentration. In addition, NO-mediated signals trigger an increase in myosin light chain phosphatase (MLCP) activity. To evaluate the mechanism of NO-induced relaxation through MLCP deinhibition, we compared time-dependent changes in Ca(2+), myosin light chain (MLC) phosphorylation and contraction to changes in phosphorylation levels of CPI-17 at Thr38, RhoA at Ser188, and MYPT1 at Ser695, Thr696 and Thr853 in response to sodium nitroprusside (SNP)-induced relaxation in denuded rabbit femoral artery. During phenylephrine (PE)-induced contraction, SNP reduced CPI-17 phosphorylation to a minimal value within 15 s, in parallel with decreases in Ca(2+) and MLC phosphorylation, followed by a reduction of contractile force having a latency period of about 15 s. MYPT1 phosphorylation at Ser695, the PKG-target site, increased concurrently with relaxation. Phosphorylation of RhoA, MYPT1 Thr696 and Thr853 differed significantly at 5 min but not within 1 min of SNP exposure. Inhibition of Ca(2+) release delayed SNP-induced relaxation while inhibition of Ca(2+) channel, BK(Ca) channel or phosphodiesterase-5 did not. Pretreatment of resting artery with SNP suppressed an increase in Ca(2+), contractile force and phosphorylation of MLC, CPI-17, MYPT1 Thr696 and Thr853 at 10 s after PE stimulation, but had no effect on phorbol ester-induced CPI-17 phosphorylation. Together, these results suggest that NO production suppresses Ca(2+) release, which causes an inactivation of PKC and rapid CPI-17 dephosphorylation as well as MLCK inactivation, resulting in rapid MLC dephosphorylation and relaxation.

Identification of DDX1 as a JC virus transcriptional control region-binding protein.

To investigate the mechanism behind JC virus (JCV) cell specificity we performed electrophoretic mobility shift assays (EMSA) using probes derived from the JCV transcriptional control region (JCV-TCR). Using nuclear extracts from the JCV-susceptible neuroblastoma cell line IMR-32, EMSA revealed a 670 kDa JCV-TCR-binding protein complex designated as #3-bp. This complex could not be detected in nuclear extracts from non-susceptible cell lines. Using column chromatographic purifi-cation and microsequencing, we identified cleavage stimulation factor (CstF) as a component of #3-bp. However, as CstF is present in many cell types, we speculated that the IMR-32-specific component(s) of #3-bp bind CstF. We performed a yeast two-hybrid assay using CstF-77 as the bait against a HeLa cDNA-subtracted IMR-32 cDNA library. This analysis detected binding between CstF-77 and the RNA helicase DDX1. Subsequently, biotinylated DNA affinity precipitation and chromatin immunoprecipitation assays also confirmed that DDX1 binds specifically to JCV-TCR. Our findings indicate that an association between DDX1 and the JCV-TCR may play a significant role in JCV infection in IMR-32 cells.

DDX1 promotes proliferation of the JC virus through transactivation of its promoter.

Recently, we demonstrated that the DEAD box protein 1 (DDX1), an RNA helicase, and the cleavage stimulation factor (CstF) form a complex that binds to the JC virus transcriptional control region (JCV-TCR). Here, we examined the function of DDX1, which is expressed at much higher levels in the JCV-susceptible cell line IMR-32 than in non-susceptible cell lines. DDX1 had no effect on the replication efficiency of JCV, but overexpression of DDX1 significantly increased transactivation of the JCV promoter. Furthermore, DDX1 enhanced the expression of JCV proteins in JCV infected cells, and knockdown of DDX1 using small interfering (si) RNA suppressed the expression of JCV proteins. Our results clearly demonstrate that DDX1 regulates proliferation of JCV in vitro through transcriptional activation.

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle.

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.