Autocrine loop between TGF-beta1 and IL-1beta through Smad3- and ERK-dependent pathways in rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) are activated during pancreatitis and promote pancreatic fibrosis by producing and secreting ECMs such as collagen and fibronectin. IL-1 has been assumed to participate in pancreatic fibrosis by activating PSCs. Activated PSCs secrete various cytokines that regulate PSC function. In this study, we have examined IL-1 secretion from culture-activated PSCs as well as its regulatory mechanism. RT-PCR and ELISA have demonstrated that PSCs express IL-1 mRNA and secrete IL-1 peptide. Inhibition of TGF-₁ activity secreted from PSCs by TGF-₁-neutralizing antibody attenuated IL-1 secretion from PSCs. Exogenous TGF-₁ increased IL-1 expression and secretion by PSCs in a dose-dependent manner. Adenovirus-mediated expression of dominant-negative (dn)Smad2/3 expression reduced both basal and TGF-₁-stimulated IL-1 expression and secretion by PSCs. Coexpression of Smad3 with dnSmad2/3 restored IL-1 expression and secretion by PSCs, which were attenuated by dnSmad2/3 expression. In contrast, coexpression of Smad2 with dnSmad2/3 did not alter them. Furthermore, inhibition of IL-1 activity secreted from PSCs by IL-1-neutralizing antibody attenuated TGF-₁ secretion from PSCs. Exogenous IL-1 enhanced TGF-₁ expression and secretion by PSCs. IL-1 activated ERK, and PD-98059, a MEK1 inhibitor, blocked IL-1 enhancement of TGF-₁ expression and secretion by PSCs. We propose that an autocrine loop exists between TGF-₁ and IL-1 in activated PSCs through Smad3- and ERK-dependent pathways. fibrosis; cytokine; chronic pancreatitis     

beta-Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of beta1 integrin signaling and mitochondrial pathway

Stimulation of -adrenergic receptors (-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits -AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with ₁ integrins. Herein we tested the hypothesis that MMP-2 impairs ₁ integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited -AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated -AR-stimulated decreases in mitochondrial membrane potential. Overexpression of ₁ integrins using adenoviruses expressing the human _1A-integrin decreased -AR-stimulated cytochrome c release and apoptosis. Overexpression of ₁ integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the ₁ integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis. matrix metalloproteinases; focal adhesion kinase; c-Jun NH₂-terminal kinase; cytochrome c     

Effect of cyclic stretch on beta1D-integrin expression and activation of FAK and RhoA

Integrins play a pivotal role in proliferation, differentiation, and survival in skeletal and cardiac myocytes. The _1D-isoform of the ₁-integrin is specifically expressed in striated skeletal muscle. However, little is known about the role and the mechanisms by which the splice variant _1D-integrin regulates myogenesis and mechanotransduction. We observed that cyclic mechanical stretch increases _1D-integrin protein levels and activates the downstream cytoskeletal signaling proteins focal adhesion kinase (FAK) and RhoA. Elimination of native _1D-integrin expression by RNA interference in immature developing myoblasts abolished stretch-induced increases in FAK phosphorylation and further downregulated RhoA activity. Blocking of _1D-integrin expression prevented myocellular fusion to form multinucleated mature myotubes. Restoration of human _1D-integrin expression in _1D-integrin-deficient cells partially restored myotube formation. The onset of myofusion also requires the generation of nitric oxide (NO). The release of NO affects cytoskeletal proteins by mediating RhoA activity and protein degradation. Our previous study demonstrated that stretch-induced NO positively modulates mechanical properties of differentiating skeletal myocytes. We found a significant decrease in NO production and apparent elastic modulus in _1D-integrin-deficient cells, suggesting signaling interactions between _1D-integrin and neuronal NO synthase to mediate mechanotransduction and myogenesis in skeletal myocytes. These results suggest that, in addition to regulating differentiation, the _1D-integrin isoform plays a critical role in the response of skeletal myoblasts to cyclic stretch by activating the downstream components of FAK and RhoA activity and affecting NO release. focal adhesion kinase; RhoA activity; mechanotransduction; skeletal myocytes     

Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction     

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle

-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of G_s and G occurs and active G_s, measured as GTP-³⁵S bound, decreases. Because intracellular Ca²⁺ is elevated in Duchenne muscular dystrophy and G_s is known to activate the dihydropyridine receptor Ca²⁺ channel, whether laminin also altered intracellular Ca²⁺ was investigated. Laminin-1 decreases active (GTP-S-bound) G_s, and the Ca²⁺ channel is inhibited by laminin-1. The laminin ₁-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C₂C₁₂ myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain     

Matrix metalloproteinases mediate beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in -adrenergic receptor (-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). -AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed -AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited -AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with ₁-integrins after -AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of ₁-integrin signaling using laminin inhibited the increased association of MMP-2 with ₁-integrins. -AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) -AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits -AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with ₁ integrins and poly-ADP-ribose-polymerase cleavage. integrins; poly-ADP-ribose-polymerase     

Control of Na+-K+-ATPase beta 1-subunit expression: role of 3'-untranslated region

Using in vitro translation and cell transfection assays, we previously demonstrated that the Na⁺-K⁺-ATPase ₁ mRNA species containing its longest 3'-untranslated region (UTR) exhibited the lowest translational efficiency. Here, employing deletions and in vivo expression assays, using direct injection of plasmids into rat ventricular myocardium, we identified a 143-nt segment located in the distal 3'-UTR of ₁ mRNA that was associated with decreased luciferase expression; interestingly, this segment contains three AUUUA motifs. Using RNA-protein binding assays and UV cross-linking of cRNA with cytosolic proteins of rat heart, we identified an 38-kDa protein that specifically bound to the cRNA encoding the 143-nt segment of ₁ mRNA 3'-UTR. Mutation of three nucleotides located in the middle region of the 143-nt segment, which was predicted to greatly disrupt a putative stem-loop structure of the cRNA in this region, was associated with reduced binding of the mutated cRNA to the protein migrating at 38 kDa. The cRNA encoding a segment of cyclooxygenase-2 mRNA 3'-UTR containing six AUUUA sequences did not bind the protein migrating at 38 kDa and did not compete with the binding of the wild-type 143-nt ₁ cRNA to the protein. The above results suggest that the 143-nt segment in the distal segment of the 3'-UTR of ₁ mRNA may play an important role in the control of ₁-subunit expression. RNA-protein binding; AUUUA sequence; plasmid expression in heart; direct myocardial injection; cardiac expression     

Ribosomal S6 kinase-1 modulates interleukin-1beta-induced persistent activation of NF-kappaB through phosphorylation of IkappaBbeta

Activation of NF-B requires the phosphorylation and degradation of its associated inhibitory proteins, IB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1 to induce persistent activation of NF-B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-B activation. In cultured rat VSMCs, IL-1 activated ERK and induced degradation of both IB and IB, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an IB/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1 decreased IB in the RSK1/IB/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced IB decrease without influencing ether ERK phosphorylation or the earlier IB degradation. By using recombinant wild-type and mutant IB proteins, both active ERK2 and RSK1 were found to directly phosphorylate IB, but only active RSK1 phosphorylated IB on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IB and contributes to the persistent activation of NF-B by IL-1. extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell     

Pathological shear stress directly regulates platelet alphaIIbbeta3 signaling

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its -subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin _IIb3 is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on _IIb3, we examined _IIb3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that -actinin, myosin heavy chain, and Syk coimmunoprecipitate with _IIb3 in resting platelets and that 120 dyn/cm² shear stress leads to their disassociation from _IIb3. Shear-induced disassociation of -actinin and myosin heavy chain from the ₃ tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to _IIb3. Syk's disassociation from ₃ is inhibited when VWF binding to either GpIb-IX-V or _IIb3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to _IIb3 but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing _IIb3 with ₃ truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates _IIb3 function and suggest that shear-induced _IIb3-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the ₃-tail. platelets; mechanoreceptor; integrin; shear stress; signal transduction     

Calcitonin gene-related peptide inhibits interleukin-1beta-induced endogenous monocyte chemoattractant protein-1 secretion in type II alveolar epithelial cells

As important multifunctional cells in the lung, alveolar epithelial type II (AEII) cells secrete numerous chemokines on various stimuli. Our previous data showed that AEII cells also express the neuropeptide calcitonin gene-related peptide (CGRP) and the proinflammatory factor interleukin (IL)-1 induces CGRP secretion in the A549 human AEII cell line. In the present study, the CGRP-1 receptor antagonist human (h)CGRP_8–37 (0.1–1 nM) greatly amplified the production of IL-1-induced monocyte chemoattractant protein (MCP)-1. The inhibition of CGRP expression by small interfering RNA significantly increased MCP-1 secretion on IL-1 stimulation. However, exogenous hCGRP (10–100 nM) suppressed IL-1-evoked MCP-1 secretion in MCP-1 promoter activity, and CGRP gene stably transfected cell clones significantly inhibited both the mRNA and protein levels of MCP-1 induced by IL-1. These data imply that AEII-derived CGRP suppressed IL-1-induced MCP-1 secretion in an autocrine/paracrine mode. Subsequent investigation revealed that CGRP inhibited IL-1-evoked NF-B activity by suppressing IB phosphorylation and degradation. Moreover, CGRP attenuated IL-1-induced reactive oxygen species (ROS) formation, the early event in proinflammatory factor signaling. We previously showed that the CGRP inhibitory effect was mediated by elevated intracellular cAMP and show here that analogs of cAMP, 8-bromoadenosine 3',5'-cyclic monophosphothioate and the Sp isomer of adenosine 3',5'-cyclic monophosphothioate, mimicked the CGRP suppressive effect on IL-1-induced ROS formation, NF-B activation, and MCP-1 secretion. Thus increased endogenous CGRP secretion in lung inflammatory disease might eliminate the excessive response by elevating the cAMP level through inhibiting the ROS-NF-B-MCP-1 pathway. reactive oxygen species; lung; inflammation     

Interleukin-1 beta induces posttranslational carboxymethylation and alterations in subnuclear distribution of lamin B in insulin-secreting RINm5F cells

We examined the effects of interleukin-1 (IL-1) treatment on the distribution and degradation of lamin B in the nuclear fraction from insulin-secreting RINm5F cells. Western blot analysis indicated that IL-1 treatment caused significant alterations in the redistribution of lamin B, specifically between the Triton X-100-soluble (membrane) and -insoluble (matrix) fractions of the nucleus. IL-1 treatment also increased the lamin carboxymethyltransferase activity and the relative abundance of the carboxymethylated lamin in the nuclear fraction. A significant increase in the relative abundance of lamin B degradation products was also observed in the nuclear fraction from the IL-1-treated cells. These findings are compatible with a measurable increase in the lamin-degrading caspase-6 activity in IL-1-treated cells. Confocal microscopic observation of IL-1-treated cells suggested a significant dissociation of lamin B from the nuclear lamina and its subsequent association with the DNA-rich elements within the nucleus. N^G-monomethyl-L-arginine, a known inhibitor of inducible nitric oxide synthetase (iNOS), markedly inhibited IL-1-induced iNOS gene expression, NO release, caspase-3 and caspase-6 activation, lamin B degradation, and loss of metabolic cell viability, indicating that the observed IL-1-induced effects on nuclear lamin B involve the intermediacy of NO. Together, our data support the hypothesis that IL-1 treatment results in significant increase in the carboxymethylation of lamin B, which would place lamin B in a strategic location for its degradation mediated by caspases. This could possibly lead to dissolution of the nuclear envelope, culminating in the demise of the effete -cell. pancreatic -cell; lamin carboxymethyltransferase; nitric oxide; nuclear matrix; caspases     

Innate immune responses of human tracheal epithelium to Pseudomonas aeruginosa flagellin, TNF-alpha, and IL-1beta

We measured innate immune responses by primary human tracheal epithelial (HTE) cells grown as confluent, pseudostratified layers during exposure to inflammatory activators on apical vs. basolateral surfaces. Apical Pseudomonas aeruginosa strain PAK (but not flagellin mutant PAK·fliC), flagellin, and flagellin + PAK·fliC activated NF-B and IL-8 expression and secretion. In contrast, HTE cells were insensitive to LPS compared to flagellin. Flagellin activated NF-B in columnar but not basal cells. IL-1 + TNF- elicited responses similar to those of flagellin. Basolateral flagellin or IL-1 + TNF- caused 1.5- to 4-fold larger responses, consistent with the fact that NF-B activation occurred in both columnar and basal cells. MyD88 (toll receptor-associated adapter), IL-1 receptor (IL1R)1, and TNF- receptor (TNFR)1 were expressed in columnar and basal cells. ZO-1 was localized to tight junctions of columnar cells but not to basal cells. We infer the following. 1) Flagellin is necessary and sufficient to trigger inflammatory responses in columnar cells during accumulation of P. aeruginosa in the airway surface liquid (ASL); columnar cells express toll-like receptor 5 and MyD88, often associated with flagellin-activated cell signaling. 2) IL-1 + TNF- in the ASL also activate columnar cells, and these cells also express IL1R1 and TNFR1. 3) Apical flagellin, IL-1, and TNF- do not activate basal cells because tight junctions between columnar cells prevent access from the apical surface to the basal cells. 4) Exposure of basolateral surfaces to inflammatory activators elicits larger responses because both columnar and basal cells are activated, likely because both cell types express receptors for flagellin, IL-1, and TNF-. toll-like receptor; nuclear factor-B; interleukin-8; tumor necrosis factor; interleukin-1     

Deglycosylation of the beta1-subunit of the BK channel changes its biophysical properties

Large-conductance Ca²⁺-activated potassium (BK) channels are composed of pore-forming -subunits and auxiliary -subunits. The -subunits are widely expressed in many cell types, whereas the -subunits are more tissue specific and influence diverse aspects of channel function. In the current study, we identified the presence of the smooth muscle-specific 1-subunit in murine colonic tissue using Western blotting. The native 1-subunits migrated in SDS-PAGE as two molecular mass bands. Enzymatic removal of N-linked glycosylations from the 1-subunit resulted in a single band that migrated at a lower molecular mass than the native 1-subunit bands, suggesting that the native 1-subunit exists in either a core glycosylated or highly glycosylated form. We investigated the functional consequence of deglycosylating the 1-subunit during inside-out single-channel recordings. During inside-out single-channel recordings, with N-glycosidase F in the pipette solution, the open probability (P_o) and mean open time of BK channels increased in a time-dependent manner. Deglycosylation of BK channels did not affect the conductance but shifted the steady-state voltage of activation toward more positive potentials without affecting slope when Ca²⁺ concentration was <1 µM. Treatment of myocytes lacking the 1-subunits of the BK channel with N-glycosidase F had no effect. These data suggest that glycosylations on the 1-subunit in smooth muscle cells can modify the biophysical properties of BK channels. peptide N-glycosidase F; large-conductance Ca²⁺-activated K⁺ channels; N-linked glycosylation; single-channel recording; auxiliary subunit     

Role of beta 1- and beta 3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes

To evaluate the physiological functions of₁-,₂-, and₃-adrenoceptors (ARs) in brownadipose tissue, the lipolytic and respiratory effects of variousadrenergic agonists and antagonists were studied in rat brownadipocytes. The -agonists stimulated both lipolysis and respiration(8-10 times above basal levels), with the following order ofpotency (concentration eliciting 50% of maximum response):CL-316243 (₃) > BRL-37344(₃) > isoproterenol (mainly₁/₂) > norepinephrine (NE; mainly₁/₂) > epinephrine (mainly₁/₂) dobutamine (₁)  procaterol (₂). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (₁ antagonist) revealed thatmore than one type of receptor mediates NE action. It is concluded fromour results that 1) NE, at low plasma levels (1-25 nM), stimulates lipolysis and respiration mainly through ₁-ARs,2) NE, at higher levels, stimulateslipolysis and respiration via both₁- and₃-ARs,3)₂-ARs play only a minor role,and 4)₃-ARs may represent thephysiological receptors for the high NE concentrations in the synapticcleft, where the high-affinity₁-ARs are presumablydesensitized. It is also suggested that lipolysis represents theflux-generating step regulating mitochondrial respiration.

     

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca²⁺-activated K⁺ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the -subunit of the BK channel (BK-), both - and ₁-subunits (BK-₁), or both - and ₄-subunits (BK-₄) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-₁ within a similar concentration range. Because methAEA could potentiate BK-, BK-₁, and BK-₄ with similar efficacy, the -subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK- and HEK-BK-₁ did not change intracellular Ca²⁺ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB₁ antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB₂ agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed. anandamide; channel opener     

Stem cell-derived Sca-1+ progenitors differentiate into smooth muscle cells, which is mediated by collagen IV-integrin alpha1/beta1/alphav and PDGF receptor pathways

Embryonic stem (ES) cells can differentiate into smooth muscle cells (SMCs) that can be used for tissue engineering and repair of damaged organs. However, little is known about the molecular mechanisms of differentiation in these cells. In the present study, we found collagen IV can promote ES cells to differentiate into stem cell antigen-1-positive (Sca-1⁺) progenitor cells and SMCs. Pretreatment of ES cells with antibodies against collagen IV significantly inhibited SMC marker expression. To further elucidate the effect of collagen IV on the induction and maintenance of SMC differentiation, Sca-1⁺ progenitor cells were isolated with magnetic beads, placed in collagen-IV-coated flasks, and cultured in differentiation medium with or without platelet-derived growth factor (PDGF)-BB for 6–90 days. Both immunostaining and fluorescence-activated cell sorter analyses revealed that the majority of these cells were positive for SMC-specific markers. Pretreatment of Sca-1⁺ progenitors with antibodies against integrin ₁, _v, and ₁, but not ₃, inhibited focal adhesion kinase (FAK) and paxillin phosphorylation and resulted in a marked inhibition of SMC differentiation. Various tyrosine kinase inhibitors, and specific siRNA for phosphatidylinositol 3-kinase (PI 3-kinase) and PDGF receptor- significantly inhibited SMC marker expression. Taken together, we demonstrate for the first time that collagen IV plays a crucial role in the early stage of SMC differentiation and that integrin (₁, ₁, and _v)-FAK-PI 3-kinase-mitogen-activated protein kinase and PDGF receptor- signaling pathways are involved in SMC differentiation. progenitor cells; extracellular matrix; growth factor receptors; platelet-derived growth factor     

Upregulation of alpha(8)beta(1)-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-beta1

Using a novel pharmacological tool with¹²⁵I-echistatin to detect integrins on the cell, we haveobserved that cardiac fibroblasts harbor five different RGD-bindingintegrins: ₈₁,₃₁, ₅₁, _v1, and _v3.Stimulation of cardiac fibroblasts by angiotensin II (ANG II) ortransforming growth factor-1 (TGF-1) resulted in an increase ofprotein and heightening by 50% of the receptor density of₈₁-integrin. The effect of ANG II wasblocked by an AT₁, but not an AT₂, receptorantagonist, or by an anti-TGF-1 antibody. ANG II and TGF-1increased fibronectin secretion, smooth muscle -actin synthesis, andformation of actin stress fibers and enhanced attachment of fibroblaststo a fibronectin matrix. The ₈- and₁-subunits were colocalized by immunocytochemistry with vinculin or ₃-integrin at focal adhesion sites.These results indicate that ₈₁-integrinis an abundant integrin on rat cardiac fibroblasts. Its positivemodulation by ANG II and TGF-1 in a myofibroblast-likephenotype suggests the involvement of₈₁-integrin in extracellularmatrix protein deposition and cardiac fibroblast adhesion.

     

Cyclooxygenase-2 is required for activated pancreatic stellate cells to respond to proinflammatory cytokines

Cyclooxygenase-2 (COX-2) mediates various inflammatory responses and is expressed in pancreatic tissue from patients with chronic pancreatitis. To examine the role of COX-2 in chronic pancreatitis, we investigated its participation in regulating functions of pancreatic stellate cells (PSCs), using isolated rat PSCs. COX-2 was expressed in culture-activated PSCs but not in freshly isolated quiescent PSCs. TGF-1, IL-1, and IL-6 enhanced COX-2 expression in activated PSCs, concomitantly increasing the expression of -smooth muscle actin (-SMA), a parameter of PSC activation. The COX-2 inhibitor NS-398 blocked culture activation of freshly isolated quiescent PSCs. NS-398 also inhibited the enhancement of -SMA expression by TGF-1, IL-1, and IL-6 in activated PSCs. These data indicate that COX-2 is required for the initiation and promotion of PSC activation. We further investigated the mechanism by which cytokines enhance COX-2 expression in PSCs. Adenovirus-mediated expression of dominant negative Smad2/3 inhibited the increase in expression of COX-2, -SMA, and collagen-1 mediated by TGF-1 in activated PSCs. Moreover, dominant negative Smad2/3 expression attenuated the expression of COX-2 and -SMA enhanced by IL-1 and IL-6. Anti-TGF- neutralizing antibody also attenuated the increase in COX-2 and -SMA expression caused by IL-1 and IL-6. IL-6 as well as IL-1 enhanced TGF-1 secretion from PSCs. These data indicate that Smad2/3-dependent pathway plays a central role in COX-2 induction by TGF-1, IL-1, and IL-6. Furthermore, IL-1 and IL-6 promote PSC activation by enhancing COX-2 expression indirectly through Smad2/3-dependent pathway by increasing TGF-1 secretion from PSCs. transforming growth factor-; interleukin; Smad; autocrine; pancreatic fibrosis     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.

     

Contribution of coupling between human myometrial beta2-adrenoreceptor and the BK(Ca) channel to uterine quiescence

The ₂-adrenergic receptor (₂-AR) and the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel have been shown, separately, to be involved in mediating uterine relaxation. Our recent studies reveal that the levels of both ₂-AR and BK_Ca channel proteins in pregnant human myometrium decrease by 50% after the onset of labor. We present direct evidence in support of a structural and functional association between the ₂-AR and the BK_Ca channel in pregnant human myometrium. Localization of both proteins is predominantly plasmalemmal, with 60% of ₂-AR colocalizing with the BK_Ca channel. Coimmunoprecipitation studies indicate that BK_Ca and ₂-AR are structurally linked by direct protein-protein interactions. Functional correlation was confirmed by experiments of human myometrial contractility in which the BK_Ca channel blocker, paxilline, significantly antagonized the relaxant effect of the ₂-AR agonist ritodrine. These novel findings provide an insight into the coupling between the ₂-AR and BK_Ca channel and may have utility in the application of this signaling cascade for therapeutic potential in the management of preterm labor. ₂-adrenergic receptor; myometrium; potassium channel; preterm labor; uterine contraction