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     

Laminin-10 and Lutheran blood group glycoproteins in adhesion of human endothelial cells

Laminin 5-chain, a constituent of laminins-10 and -11, is expressed in endothelial basement membranes. In this study we evaluated the roles of 5 laminins and Lutheran blood group glycoproteins (Lu), recently identified receptors of the laminin 5-chain, in the adhesion of human dermal microvascular and pulmonary artery endothelial cells. Field emission scanning electron microscopy and immunohistochemistry showed that the endothelial cells spread on laminin-10 and formed fibronectin-positive fibrillar adhesion structures. Immunoprecipitation results suggested that the cells produced fibronectin, which they could use as adhesion substratum, during the adhesion process. When the protein synthesis during the adhesion was inhibited with cycloheximide, the formation of fibrillar adhesions on laminin-10 was abolished, suggesting that laminin-10 does not stimulate the formation of any adhesion structures. Northern and Western blot analyses showed that the cells expressed M_r 78,000 and 85,000 isoforms of Lu. Quantitative cell adhesion assays showed that in the endothelial cell adhesion to laminin-10, Lu acted in concert with integrins ₁ and _v3, whereas in the adhesion to laminin-10/11, Lu and integrin ₁ were involved. In the cells adhering to the 5 laminins, Lu and the integrins showed uniform cell surface distribution. These findings indicate that 5 laminins stimulate endothelial cell adhesion but not the formation of fibrillar or focal adhesions. Lu mediates the adhesion of human endothelial cells to 5 laminins in collaboration with integrins ₁ and _v3. integrin; cycloheximide     

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     

Modulation of alpha7-integrin-mediated adhesion and expression by platelet-derived growth factor in vascular smooth muscle cells

We showed previously that the expression of ₇-integrin in aortic vascular smooth muscle cells (VSMC) is enhanced in a rat model of atherosclerosis. In the present study, we investigated the effects of platelet-derived growth factor (PDGF) on ₇-integrin expression and VSMC adhesion and migration. Expression of the ₇-integrin gene was determined by real-time RT-PCR, whereas protein levels were determined by fluorescence-activated cell sorting analysis. PDGF increased ₇ cell surface protein expression (12 and 24 h: 3.3 ± 0.8- and 3.6 ± 0.4-fold, P < 0.05 vs. control) and mRNA levels (24 h: 3.1-fold, P < 0.05 vs. control) in a time-dependent manner. Actinomycin D and cycloheximide attenuated PDGF-induced increases in ₇-integrin, indicating the involvement of de novo mRNA and protein synthesis. Treatment with the MAPK inhibitors PD-98059, SP-600125, and SB-203580 attenuated PDGF-induced increases in mRNA. In contrast, PD-98059 and SP-600125, but not SB-203580, attenuated PDGF-induced increases in cell surface protein levels. PDGF-treated VSMC adhered to laminin more efficiently (42 ± 6% increase, P < 0.01), and this increase was partially inhibited by anti-₇-integrin function-blocking antibody. However, PDGF did not alter migration on laminin, and there was no effect of the anti-₇-integrin function-blocking antibody on basal or PDGF-stimulated migration. Immunofluorescence imaging revealed an increase in ₇-integrin distribution along the stress fibers. Together, these observations indicate that PDGF enhances ₇-integrin expression in VSMC and promotes ₇-integrin-mediated adhesion to laminin. vascular injury; laminin; mitogen-activated protein kinase     

Protein kinase C{varepsilon} contributes to regulation of the sarcolemmal Na+-K+ pump

A reduction in angiotensinII (ANG II) in vivo by treatment of rabbits with theangiotensin-converting enzyme inhibitor, captopril, increasesNa⁺-K⁺ pump current (I_p)of cardiac myocytes. This increase is abolished by exposure of myocytesto ANG II in vitro. Because ANG II induces translocation of the-isoform of protein kinase C (PKC), we examined whether thisisozyme regulates the pump. We treated rabbits with captopril, isolatedmyocytes, and measured I_p of myocytes voltageclamped with wide-tipped patch pipettes. I_p ofmyocytes from captopril-treated rabbits was larger thanI_p of myocytes from controls. ANG II superfusionof myocytes from captopril-treated rabbits decreasedI_p to levels similar to controls. Inclusion ofPKC-specific blocking peptide in pipette solutions used to perfusethe intracellular compartment abolished the effect of ANG II. Inclusionof RACK, a PKC-specific activating peptide, in pipettesolutions had an effect on I_p that was similarto that of ANG II. There was no additive effect of ANG II andRACK. We conclude that PKC regulates the sarcolemmalNa⁺-K⁺ pump.

     

Beta-subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae

Previous studies showed the presence of a significant fraction of Na⁺-K⁺-ATPase -subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na⁺-K⁺-ATPase with its signaling partners. Because both - and -subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na⁺-K⁺-ATPase, we have examined the relative distribution of two major subunit isoforms (₁ and ₁) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na₂CO₃ or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30–40% of ₁-subunits and 80–90% of ₁-subunits. Use of Na₂CO₃ was shown to inactivate Na⁺-K⁺-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained 75% of total myocyte Na⁺-K⁺-ATPase activity. Sealed isolated caveolae exhibited active Na⁺ transport. Confocal microscopy supported the presence of ,-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na⁺-K⁺ fluxes, and the sites where the pumping and signaling functions of Na⁺-K⁺-ATPase are integrated. Preferential concentration of ₁-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of ₁ and ₁ in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na⁺-K⁺-ATPase in cardiac caveolae. cardiac myocyte; caveolin; oligomer; ouabain; sodium pump     

肝癌细胞-胞外基质粘附性与粘附识别序列的相关性

以微管吸吮技术研究了人肝癌细胞在ＩＶ型胶原／层粘连蛋白（ＬＮ）／纤维连结蛋白（ＦＮ）裱衬表面的粘附性。进一步,用四种人工合成肽精－甘－天冬－丝（ＲＧＤＳ）、甘－精－甘－天冬－苏－脯ＧＲＧＤＴＰ）、酪－异亮－甘－丝－精（ＹＩＧＳＲ０和半胱－天冬－脯－甘－酪－异亮－甘－丝－精（ＣＤＰＧＹＩＧＳＲ）研究了肝癌细胞粘附性对两种粘附识别序列ＲＧＤ和ＹＩＧＳＲ的依赖性。为了归纳和整理实验结果,根据竞争性抑制的     

Embryonic heart mesenchymal cell migration in laminin

Laminin, a large glycoprotein and major component of basement membranes, influences cell adhesion, migration, morphology, and differentiation. A peptide sequence, YIGSR, from the B1 chain of laminin has been found to correspond to an active site for cell adhesion. We report here that cardiac mesenchymal cells migrate vigorously within three-dimensional gels of laminin and that the YIGSR peptide will completely abolish this migratory activity. In contrast, migration of the mesenchymal cells into three-dimensional gels composed of collagen or collagen + laminin is not effected by YIGSR or other peptides (GRGDS, GRGDTP) reported to mediate cellular adhesion.     

Regulation of the cardiac L-type Ca2+ channel by the actin-binding proteins alpha-actinin and dystrophin

Theactin-binding proteins dystrophin and -actinin are members of afamily of actin-binding proteins that may link the cytoskeleton tomembrane proteins such as ion channels. Previous work demonstrated thatthe activity of Ca²⁺ channels can be regulated by agentsthat disrupt or stabilize the cytoskeleton. In the present study, weemployed immunohistochemical and electrophysiological techniques toinvestigate the potential regulation of cardiac L-type Ca²⁺channel activity by dystrophin and -actinin in cardiac myocytes andin heterologous cells. Both actin-binding proteins were found tocolocalize with the Ca²⁺ channel in mouse cardiac myocytesand to modulate channel function. Inactivation of the Ca²⁺channel in cardiac myocytes from mice lacking dystrophin(mdx mice) was reduced compared with that in wild-typemyocytes, voltage dependence of activation was shifted by 5 mV to morepositive potentials, and stimulation by the -adrenergic pathway andthe dihydropyridine agonist BAY K 8644 was increased. Furthermore, heterologous coexpression of the Ca²⁺ channel with muscle,but not nonmuscle, forms of -actinin was also found to reduceinactivation. As might be predicted from a reduction ofCa²⁺ channel inactivation, a prolonging of the mouseelectrocardiogram QT was observed in mdx mice. These resultssuggest a combined role for dystrophin and -actinin in regulatingthe activity of the cardiac L-type Ca²⁺ channel and apotential mechanism for cardiac dysfunction in Duchenne and Beckermuscular dystrophies.

     

Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes

The Na⁺/K⁺-ATPase (NKA) is the main route for Na⁺ extrusion from cardiac myocytes. Different NKA -subunit isoforms are present in the heart. NKA-1 is predominant, although there is a variable amount of NKA-2 in adult ventricular myocytes of most species. It has been proposed that NKA-2 is localized mainly in T-tubules (TT), where it could regulate local Na⁺/Ca²⁺ exchange and thus cardiac myocyte Ca²⁺. However, there is controversy as to where NKA-1 vs. NKA-2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (I_Pump) measurements and the different ouabain sensitivity of NKA-1 (low) and NKA-2 (high) in rat heart. Ouabain-dependent I_Pump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-2, K_1/2 = 0.38 ± 0.16 µM) that accounts for 29.5 ± 1.3% of I_Pump and a low-affinity isoform (NKA-1, K_1/2 = 141 ± 17 µM) that accounts for 70.5% of I_Pump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced I_Pump density from 1.24 ± 0.05 to 1.02 ± 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-2 accounted for only 18.2 ± 1.1% of I_Pump. Thus, 63% of I_Pump generated by NKA-2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-2/NKA-1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-2 is 4.5 times higher in the T-tubules vs. ESL, whereas NKA-1 is almost uniformly distributed between the TT and ESL. T-tubules; Na⁺/K⁺ pump current; ouabain; external sarcolemma; detubulation     

The prolyl hydroxylase oxygen-sensing pathway is cytoprotective and allows maintenance of mitochondrial membrane potential during metabolic inhibition

The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase domain (PHD)-containing enzymes, whose reduction of activity initiate a hypoxic signal cascade. In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes. PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels in glucose-free medium containing cyanide. The addition of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) caused a decline of ATP levels that was indistinguishable between control and PHI-treated myocytes. Despite the comparable levels of ATP depletion, PHI-preconditioned myocytes remained significantly protected. As expected, mitochondrial membrane potential (_mito) collapses in control myocytes during cyanide and 2-DG treatment and it fails to completely recover upon washout. In contrast, _mito is partially maintained during metabolic inhibition and recovers completely on washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse _mito in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of _mito in PHI-treated myocytes, whereas a role for the "reverse mode" operation of the F₁F₀-ATP synthase was ruled out. Further examination of mitochondrial function revealed that PHI treatment downregulated basal oxygen consumption to only 15% that of controls. Oxygen consumption rates, although initially lower in PHI-preconditioned myocytes, recovered completely upon removal of metabolic poisons, while reaching only 22% of preinsult levels in control myocytes. We conclude that PHD oxygen-sensing mechanism directs multiple compensatory changes in the cardiomyocyte, which include a low-respiring mitochondrial phenotype that is remarkably protected against metabolic insult. fumarate; hibernation; cardioprotection; anaplerotic     

beta-amyloid-induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1

In patients withamyloid -related cerebrovascular disorders, e.g., Alzheimer'sdisease, one finds increased deposition of amyloid peptide (A) andincreased presence of monocyte/microglia cells in the brain. However,relatively little is known of the role of A in the trafficking ofmonocytes across the blood-brain barrier (BBB). Our studies show thatinteraction of A_1-40 with monolayer of human brainendothelial cells results in augmented adhesion and transendothelialmigration of monocytic cells (THP-1 and HL-60) and peripheral bloodmonocytes. The A-mediated migration of monocytes was inhibited byantibody to A receptor (RAGE) and platelet endothelial cell adhesionmolecule (PECAM-1). Additionally, A-induced transendothelialmigration of monocytes were inhibited by protein kinase C inhibitor andaugmented by phosphatase inhibitor. We conclude that interaction ofA with RAGE expressed on brain endothelial cells initiates cellularsignaling leading to the transendothelial migration of monocytes. Wesuggest that increased diapedesis of monocytes across the BBB inresponse to A present either in the peripheral circulation or in thebrain parenchyma may play a role in the pathophysiology of A-relatedvascular disorder.

     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Skeletal muscle LIM protein 1 (SLIM1/FHL1) induces alpha 5 beta 1-integrin-dependent myocyte elongation

Skeletal muscle LIM protein 1 (SLIM1/FHL1) contains four and a half LIM domains and is highly expressed in skeletal and cardiac muscle. Elevated SLIM1 mRNA expression has been associated with postnatal skeletal muscle growth and stretch-induced muscle hypertrophy in mice. Conversely, SLIM1 mRNA levels decrease during muscle atrophy. Together, these observations suggest a link between skeletal muscle growth and increased SLIM1 expression. However, the precise function of SLIM1 in skeletal muscle, specifically the role of SLIM1 during skeletal muscle differentiation, is not known. This study investigated the effect of increased SLIM1 expression during skeletal muscle differentiation. Western blot analysis showed an initial decrease followed by an increase in SLIM1 expression during differentiation. Overexpression of SLIM1 in Sol8 or C₂C₁₂ skeletal muscle cell lines, at levels observed during hypertrophy, induced distinct effects in differentiating myocytes and undifferentiated reserve cells, which were distinguished by differential staining for two markers of differentiation, MyoD and myogenin. In differentiating skeletal myocytes, SLIM1 overexpression induced hyperelongation, which, by either plating cells on poly-L-lysine or using a series of peptide blockade experiments, was shown to be specifically dependent on ligand binding to the ₅₁-integrin, whereas in reserve cells, SLIM1 overexpression induced the formation of multiple cytoplasmic protrusions (branching), which was also integrin mediated. These results suggest that SLIM1 may play an important role during the early stages of skeletal muscle differentiation, specifically in ₅₁-integrin-mediated signaling pathways. myoblast; proteins and differentiation     

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     

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     

Enhancement of L-type Ca(2+) current from neonatal mouse ventricular myocytes by constitutively active PKC-betaII

The cardiacL-type calcium current (I_Ca) can be modified byactivation of protein kinase C (PKC). However, the effect of PKC activation on I_Ca is still controversial. Somestudies have shown a decrease in current, whereas other studies havereported a biphasic effect (an increase followed by a decrease incurrent or vice versa). A possible explanation for the conflictingresults is that several isoforms of PKC with opposing effects onI_Ca were activated simultaneously. Here, weexamined the influence of a single PKC isoform (PKC-II) on L-typecalcium channels in isolation from other cardiac isoforms, using atransgenic mouse that conditionally expresses PKC-II. Ventricularcardiac myocytes were isolated from newborn mice and examined forexpression of the transgene using single cell RT-PCR afterI_Ca recording. Cells expressing PKC-II showeda twofold increase in nifedipine-sensitive I_Ca. The PKC-II antagonist LY-379196 returned I_Caamplitude to levels found in non-PKC-II-expressing myocytes. Theincrease in I_Ca was independent ofCa_v1.2-subunit mRNA levels as determined by quantitativeRT-PCR. Thus these data demonstrate that PKC- is a potent modulatorof cardiac L-type calcium channels and that this specific isoformincreases I_Ca in neonatal ventricular myocytes.

     

Regulation and distribution of MAdCAM-1 in endothelial cells in vitro

Mucosal addressin cell adhesion molecule-1(MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein thatregulates lymphocyte trafficking to Peyer's patches and lymph nodes.Although it is widely agreed that MAdCAM-1 induction is involved inchronic gut inflammation, few studies have investigated regulation ofMAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)- using RT-PCR, blotting, adhesion, and immunofluorescence. TNF- induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38mitogen-activated protein kinase (MAPK), and nuclear factor(NF)-B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1is regulated via MAPKs, we examined mitogen/extracellularsignal-regulated kinase (MEK)-1/2 activation in SVEC. We found thatMEK-1/2 is activated by TNF- within minutes and is dependent on TKand p42/44 MAPKs. Similarly, TNF- activated NF-B through TK,p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was alsoshown to be frequently distributed to endothelial junctions both invitro and in vivo. Cytokines like TNF- stimulate MAdCAM-1 inhigh endothelium via TK, p38, p42/22 MAPKs, and NF-B/PARP.MAdCAM-1 expression requires NF-B translocation through both directp42/44 and indirect p38 MAPK pathways in high endothelial cells.

     

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G_q-coupled P2Y₂ receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of - and -actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of - or -actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca²⁺-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis. lung; mucus; exocytosis     

Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells

Overexpression of aconstitutively active mutant of the mitogen-activated protein kinasekinase MEK1 (caMEK1) in epithelial Madin-Darby canine kidney (MDCK)-C7cells disrupts morphogenesis, induces an invasive phenotype, and isassociated with a reduced rate of cell proliferation. The role ofcell-cell adhesion molecules and cell cycle proteins in theseprocesses, however, has not been investigated. We now report loss ofE-cadherin expression as well as a marked reduction of - and-catenin expression in transdifferentiated MDCK-C7 cells stablyexpressing caMEK1 (C7caMEK1) compared with epithelial mock-transfectedMDCK-C7 (C7Mock1) cells. At least part of the remaining -catenin wascoimmunoprecipitated with -catenin, whereas no E-cadherin wasdetected in -catenin immunoprecipitates. In both cell types, theproteasome-specific protease inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to atime-dependent accumulation of -catenin, including the appearance ofhigh-molecular-weight -catenin species. Quiescent as well asserum-stimulated C7caMEK1 cells showed a higher cyclin D expressionthan epithelial C7Mock1 cells. The MEK inhibitor U-0126 inhibitedextracellular signal-regulated kinase phosphorylation and cyclin Dexpression in C7caMEK1 cells and almost abolished their already reducedcell proliferation rate. We conclude that the transdifferentiated andinvasive phenotype of C7caMEK1 cells is associated with a diminishedexpression of proteins involved in cell-cell adhesion. Although-catenin expression is reduced, C7caMEK1 cells show a higherexpression of U-0126-sensitive cyclin D protein.