Three-dimensional tissue structure affects sensitivity of fibroblasts to TGF-beta 1

Transforming growth factor-(TGF-) is known to induce -smooth muscle actin (-SMA) infibroblasts and is supposed to play a role in myofibroblastdifferentiation and tumor desmoplasia. Our objective was to elucidatethe impact of TGF-1 on -SMA expression in fibroblasts in athree-dimensional (3-D) vs. two-dimensional (2-D) environment. Inmonolayer culture, all fibroblast cultures responded in a similarfashion to TGF-1 with regard to -SMA expression. In fibroblastspheroids, -SMA expression was reduced and induction by TGF-1 washighly variable. This difference correlated with a differentialregulation in the TGF- receptor (TGFR) expression, in particularwith a reduction in TGF-RII in part of the fibroblast types. Ourdata indicate that 1) sensitivity to TGF-1-induced -SMA expression in a 3-D environment is fibroblast-type specific, 2) fibroblast type-independent regulatory mechanisms, suchas a general reduction/loss in TGF-RIII, contribute to an altered TGFR expression profile in spheroid compared with monolayer culture, and 3) fibroblast type-specific alterations in TGFR typesI and II determine the sensitivity to TGF-1-induced -SMAexpression in the 3-D setting. We suggest that fibroblasts that can beinduced by TGF-1 to produce -SMA in spheroid culture reflect a"premyofibroblastic" phenotype.

     

Differential expression of TGF-beta isoforms by normal and inflammatory bowel disease intestinal myofibroblasts

First publishedSeptember 5, 2001; 10.1152/ajpcell. 00048.2001.Intestinalstrictures are frequent in Crohn's disease but not ulcerative colitis.We investigated the expression of transforming growth factor (TGF)-isoforms by isolated and cultured primary human intestinalmyofibroblasts and the responsiveness of these cells and intestinalepithelial cells to TGF- isoforms. Normal intestinal myofibroblastsreleased predominantly TGF-₃ and ulcerative colitismyofibroblasts expressed both TGF-₁ andTGF-₃, whereas in myofibroblast cultures from fibroticCrohn's disease tissue, there was significantly lower expression ofTGF-₃ but enhanced release of TGF-₂.These distinctive patterns of TGF- isoform release were sustainedthrough several myofibroblast passages. Proliferation of Crohn'sdisease myofibroblasts was significantly greater than that ofmyofibroblasts derived from normal and ulcerative colitis tissue. Incontrast to cells from normal and ulcerative colitis tissue,neutralization of the three TGF- isoforms did not affect theproliferation of Crohn's disease intestinal myofibroblasts. Studies onthe effect of recombinant TGF- isoforms on epithelial restitutionand proliferation suggest that TGF-₂ may be the least effective of the three isoforms in intestinal wound repair. In conclusion, the enhanced release of TGF-₂ but reducedexpression of TGF-₃ by Crohn's disease intestinalmyofibroblasts, together with their enhanced proliferative capacity,may lead to the development of intestinal strictures.

     

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.

     

The gamma-subunit of ENaC is more important for channel surface expression than the beta-subunit

The amiloride-sensitiveepithelial sodium channel (ENaC) plays a critical role in fluid andelectrolyte homeostasis and is composed of three homologous subunits:, , and . Only heteromultimeric channels made of ENaCare efficiently expressed at the cell surface, resulting in maximallyamiloride-sensitive currents. To study the relative importance ofvarious regions of the - and -subunits for the expression offunctional ENaC channels at the cell surface, we constructedhemagglutinin (HA)-tagged --chimeric subunits composed of -and -subunit regions and coexpressed them with HA-tagged - and-subunits in Xenopus laevis oocytes. The whole cellamiloride-sensitive sodium current (I_ami) andsurface expression of channels were assessed in parallel using thetwo-electrode voltage-clamp technique and a chemiluminescence assay.Because coexpression of ENaC resulted in largerI_ami and surface expression compared withcoexpression of ENaC, we hypothesized that the -subunit ismore important for ENaC trafficking than the -subunit. Usingchimeras, we demonstrated that channel activity is largely preservedwhen the highly conserved second cysteine rich domains (CRD2) of the- and -subunits are exchanged. In contrast, exchanging the wholeextracellular loops of the - and the -subunits largely reducedENaC currents and ENaC expression in the membrane. This indicates thatthere is limited interchangeability between molecular regions of thetwo subunits. Interestingly, our chimera studies demonstrated that theintracellular termini and the two transmembrane domains of ENaC aremore important for the expression of functional channels at the cellsurface than the corresponding regions of ENaC.

     

Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells

The aim of thisstudy was to identify fibrogenic mediators stimulatingactivation, proliferation, and/or matrix synthesis of rat pancreaticstellate cells (PSC). PSC were isolated from the pancreas of normalWistar rats and from rats with cerulein pancreatitis. Cell activationwas demonstrated by immunofluorescence microscopy of smooth muscle-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin,and transforming growth factor (TGF)-₁. Proliferationwas measured by bromodeoxyuridine incorporation. Matrix synthesis wasdemonstrated on the protein and mRNA level. Within a few days inprimary culture, PSC changed their phenotype from fat-storing toSMA-positive myofibroblast-like cells expressing platelet-derivedgrowth factor (PDGF) - and PDGF -receptors. TGF-₁and tumor necrosis factor (TNF)- accelerated the change in thecells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basicfibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-₁ (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF- (1.87 ± 0.19-fold). As shownby RT-PCR, PSC express predominantly the splice variant EIII-A offibronectin. Immunofluorescence microscopy and Northern blot confirmedthat in particular bFGF and TGF-₁ stimulated thesynthesis of fibronectin and collagens type I and III. In conclusion,our data demonstrate that 1) TGF-₁ andTNF- accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF,TGF-₁, PDGF, and, to a lesser extent, TGF- stimulateextracellular matrix synthesis of cultured rat PSC.

     

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.

     

Role of alpha(v)beta(3)-integrin in TNF-alpha-induced endothelial cell migration

Tumor necrosis factor- (TNF-), oneof the major inflammatory cytokines, is known to influence endothelialcell migration. In this study, we demonstrate that exposure of calfpulmonary artery endothelial cells to TNF- caused an increase in theformation of membrane protrusions and cell migration. Fluorescencemicroscopy revealed an increase in _v3focal contacts but a decrease in ₅₁ focalcontacts in TNF--treated cells. In addition, both cell-surface andtotal cellular expression of _v3-integrinsincreased significantly, whereas the expression of₅₁-integrins was unaltered. Only focalcontacts containing _v3- but not₅₁-integrins were present in membraneprotrusions of cells at the migration front. In contrast, robust focalcontacts containing ₅₁-integrins were present in cells behind the migration front. A blocking antibody to_v3, but not a blocking antibody to₅-integrins, significantly inhibited TNF--inducedcell migration. These results indicate that in response to TNF-,endothelial cells may increase the activation and ligation of_v3 while decreasing the activation andligation of ₅₁-integrins to facilitatecell migration, a process essential for vascular wound healing and angiogenesis.

     

Rapid and dynamic regulation of TGF-beta receptors on blood vessels and fibroblasts during ischemia-reperfusion injury

Thepathophysiological mechanisms involved in ischemia-reperfusioninjury are poorly understood. Although transforming growth factor(TGF)- has been shown to provide protection againstischemia-reperfusion injury in different organ systems, littleis known about the regulation of TGF- action during this process.Here we analyzed the effect of ischemia and reperfusion on theexpression of TGF- and its receptors in vivo with a pig skin flapmodel. Analysis of unoperated skin, nonischemic control flap,ischemic flap, and reperfused flap by immunohistochemistryindicates that ischemia and reperfusion result in rapid anddynamic regulation of type I, II, and III TGF- receptors andTGF-1 in a cell type-specific manner. Furthermore, hypoxiaupregulates type II TGF- receptor mRNA in skin fibroblasts inculture. Together, our results reveal that TGF- receptors andTGF-1 are markedly increased under acute ischemic conditions in the blood vessels and fibroblasts of the skin. We conclude thatTGF- action is enhanced under ischemic conditions and that it may represent an adaptive response to ischemic injury. The augmented TGF- responsiveness may be a critical determinant of theprotective effect of TGF- during ischemia-reperfusion injury.

     

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Na(+) channels

Investigation of the role ofindividual protein kinase C (PKC) isozymes in the regulation ofNa⁺ channels has been largely limited by the lack ofisozyme-selective modulators. Here we used a novel peptide-specificactivator (V1-7) of PKC and other peptide isozyme-specificinhibitors in addition to the general PKC activator phorbol12-myristate 13-acetate (PMA) to dissect the role of individual PKCs inthe regulation of the human cardiac Na⁺ channel hH1,heterologously expressed in Xenopus oocytes. Peptides wereinjected individually or in combination into the oocyte. Whole cellNa⁺ current (I_Na) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited I_Na by 31 ± 5% and 44 ± 8% (at 20 mV), respectively. These effects were not seen with thescrambled peptide for V1-7 (100 nM) or the PMA analog4-phorbol 12,13-didecanoate (100 nM). However, V1-7-and PMA-induced I_Na inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced I_Na inhibition was not altered by100 nM peptide-specific inhibitors for -, -, -, or PKC. PMAand V1-7 induced translocation of PKC from soluble toparticulate fraction in Xenopus oocytes. This translocationwas antagonized by V1-2. In native rat ventricular myocytes,PMA and V1-7 also inhibited I_Na; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na⁺channels by PKC isozyme.

     

Expression of the TGF-beta receptor gene and sensitivity to growth inhibition following polyamine depletion

Our previous studieshave shown that inhibition of polyamine biosynthesis increases thesensitivity of intestinal epithelial cells to growth inhibition inducedby exogenous transforming growth factor- (TGF-). This study wentfurther to determine whether expression of the TGF- receptor genesis involved in this process. Studies were conducted in the IEC-6 cellline, derived from rat small intestinal crypt cells. Administration of-difluoromethylornithine (DFMO), a specific inhibitor of ornithinedecarboxylase (the rate-limiting enzyme for polyamine synthesis), for 4 and 6 days depleted cellular polyamines putrescine, spermidine, andspermine in IEC-6 cells. Polyamine depletion by DFMO increased levelsof the TGF- type I receptor (TGF-RI) mRNA and protein but had noeffect on the TGF- type II receptor expression. The inducedTGF-RI expression after polyamine depletion was associated with anincreased sensitivity to growth inhibition induced by exogenous TGF-but not by somatostatin. Extracellular matrix laminin inhibited IEC-6cell growth without affecting the TGF- receptor expression. Lamininconsistently failed to induce the sensitivity of TGF--mediatedgrowth inhibition. In addition, decreasing TGF-RI expression bytreatment with retinoic acid not only decreased TGF--mediated growthinhibition in normal cells but also prevented the increased sensitivityto exogenous TGF- in polyamine-deficient cells. These resultsindicate that 1) depletion of cellular polyamines by DFMOincreases expression of the TGF-RI gene and 2) increasedTGF-RI expression plays an important role in the process throughwhich polyamine depletion sensitizes intestinal epithelial cells togrowth inhibition induced by TGF-.

     

Laminins and TGF-beta maintain cell polarity and functionality of human gastric glandular epithelium

The human gastric glandularepithelium produces a gastric lipase enzyme (HGL) that plays animportant role in digestion of dietary triglycerides. To assess theinvolvement of extracellular matrix components and transforming growthfactor-1 (TGF-1) in the regulation of this enzymic function,normal gastric epithelial cells were cultured on collagen type I,Matrigel, and laminins (LN)-1 and -2 with or without TGF-1.Epithelial morphology and HGL expression were evaluated usingmicroscopy techniques, enzymic assays, Western blot, Northernhybridization, and RT-PCR. A correlation was observed between the cellpolarity status and the level of HGL expression. TGF-1 alone orindividual matrix components stimulated cell spreading and caused adownfall of HGL activity and mRNA. By contrast, Matrigel preserved themorphological features of differentiated epithelial cells andmaintained HGL expression. The combination of LNs with TGF-1 (twoconstituents of Matrigel) exerted similar beneficial effects onepithelial cell polarity and evoked a 10-fold increase of HGL levelsthat was blunted by a neutralizing antibody against the₂-integrin subunit and by mitogen-activated proteinkinase (MAPK) inhibitors PD-98059 (p42/p44) or SB-203580 (p38). Thisinvestigation demonstrates for the first time that a powerful synergismbetween a growth factor and basement membrane LNs positively influencescell polarity and functionality of the human gastric glandularepithelium through an activation of the₂₁-integrin and effectors of two MAPK pathways.

     

High glucose and endothelial cell growth: novel effects independent of autocrine TGF-beta 1 and hyperosmolarity

Human endothelial cells wereexposed to 5 mM glucose (control), 25 mM (high) glucose, or osmoticcontrol for 72 h. TGF-1 production, cell growth, death, andcell cycle progression, and the effects of TGF-1 and TGF-neutralization on these parameters were studied. High glucose andhyperosmolarity increased endothelial TGF-1 secretion(P < 0.0001) and bioactivity (P < 0.0001). However, high glucose had a greater effect on reducingendothelial cell number (P < 0.001) and increasingcellular protein content (P < 0.001) than the osmoticcontrol. TGF- antibody only reversed the antiproliferative andhypertrophic effects of high glucose. High glucose altered cell cycleprogression and cyclin-dependent kinase inhibitor expressionindependently of hyperosmolarity. High glucose increased endothelialcell apoptosis (P < 0.01), whereashyperosmolarity induced endothelial cell necrosis (P < 0.001). TGF- antibody did not reverse the apoptotic effectsobserved with high glucose. Exogenous TGF-1 mimicked the increased Sphase delay but not endoreduplication observed with high glucose. High glucose altered endothelial cell growth, apoptosis, and cellcycle progression. These growth effects occurred principally via aTGF-1 autocrine pathway. In contrast, apoptosis andendoreduplication occurred independently of this cytokine and hyperosmolarity.

     

PPAR-gamma ligands modulate effects of LPS in stimulated rat synovial fibroblasts

This work demonstrated the constitutive expressionof peroxisome proliferator-activated receptor (PPAR)- and PPAR-in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR- expression induced by 10 µg/ml lipopolysaccharide (LPS) was observed, whereas PPAR- mRNA expression was not modified. 15-Deoxy-^12,14-prostaglandin J₂(15d-PGJ₂) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (80%) and inducible nitric oxide synthase (iNOS) mRNA expression (80%), whereas troglitazone (10 µM) only inhibited iNOS mRNA expression (50%). 15d-PGJ₂ decreasedLPS-induced interleukin (IL)-1 (25%) and tumor necrosis factor(TNF)- (40%) expression. Interestingly, troglitazone stronglydecreased TNF- expression (50%) but had no significant effect onIL-1 expression. 15d-PGJ₂ was able to inhibitDNA-binding activity of both nuclear factor (NF)-B and AP-1.Troglitazone had no effect on NF-B activation and was shown toincrease LPS-induced AP-1 activation. 15d-PGJ₂ andtroglitazone modulated the expression of LPS-induced iNOS, COX-2, andproinflammatory cytokines differently. Indeed, troglitazone seems tospecifically target TNF- and iNOS pathways. These results offer newinsights in regard to the anti-inflammatory potential of the PPAR-ligands and underline different mechanisms of action of15d-PGJ₂ and troglitazone in synovial fibroblasts.

     

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.

     

Alterations in airway ion transport in NKCC1-deficient mice

Airways of Na⁺-K⁺-2Cl(NKCC1)-deficient mice (/) were studied in Ussing chambers todetermine the role of the basolateral NKCC1 in transepithelial anionsecretion. The basal short-circuit current (I_sc)of tracheae and bronchi from adult mice did not differ betweenNKCC1/ and normal mice, whereas NKCC1/ tracheae from neonatalmice exhibited a significantly reduced basalI_sc. In normal mouse tracheae, sensitivity tothe NKCC1 inhibitor bumetanide correlated inversely with the age of themouse. In contrast, tracheae from NKCC1/ mice at all ages wereinsensitive to bumetanide. The anion secretory response to forskolindid not differ between normal and NKCC1/ tissues. However, whenlarger anion secretory responses were induced with UTP, airways fromthe NKCC1/ mice exhibited an attenuated response. Ion substitutionand drug treatment protocols suggested that HCOsecretion compensated for reduced Cl secretion inNKCC1/ airway epithelia. The absence of spontaneous airway diseaseor pathology in airways from the NKCC1/ mice suggests that theNKCC1 mutant mice are able to compensate adequately for absence of theNKCC1 protein.

     

Polyamines regulate beta-catenin tyrosine phosphorylation via Ca(2+) during intestinal epithelial cell migration

Polyaminesare essential for early mucosal restitution that occurs by epithelialcell migration to reseal superficial wounds after injury. Normalintestinal epithelial cells are tightly bound in sheets, but they needto be rapidly disassembled during restitution. -Catenin is involvedin cell-cell adhesion, and its tyrosine phosphorylation causesdisassembly of adhesion junctions, enhancing the spreading of cells.The current study determined whether polyamines are required for thestimulation of epithelial cell migration by altering -catenintyrosine phosphorylation. Migration of intestinal epithelial cells(IEC-6 line) after wounding was associated with an increase in-catenin tyrosine phosphorylation, which decreased the bindingactivity of -catenin to -catenin. Polyamine depletion by-difluoromethylornithine reduced cytoplasmic free Ca²⁺concentration ([Ca²⁺]_cyt), preventedinduction of -catenin phosphorylation, and decreased cell migration.Elevation of [Ca²⁺]_cyt induced by theCa²⁺ ionophore ionomycin restored -cateninphosphorylation and promoted migration in polyamine-deficient cells.Decreased -catenin phosphorylation through the tyrosine kinaseinhibitor herbimycin-A or genistein blocked cell migration, which wasaccompanied by reorganization of cytoskeletal proteins. These resultsindicate that -catenin tyrosine phosphorylation plays a criticalrole in polyamine-dependent cell migration and that polyamines induce-catenin tyrosine phosphorylation at least partially through[Ca²⁺]_cyt.

     

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.

     

GSK-3beta negatively regulates skeletal myotube hypertrophy

Todetermine whether changes in glycogen synthase kinase-3 (GSK-3)phosphorylation contribute to muscle hypertrophy, we delineated theeffects of GSK-3 activity on C₂C₁₂ myotubesize. We also examined possible insulin-like growth factor I (IGF-I) signaling of NFAT (nuclear factors of activated T cells)-inducible geneactivity and possible modulation of NFAT activation by GSK-3. Application of IGF-I (250 ng/ml) or LiCl (10 mM) alone (i.e., bothinhibit GSK-3 activity) increased the area ofC₂C₁₂ myotubes by 80 and 85%, respectively.The application of IGF-I (250 ng/ml) elevated GSK-3 phosphorylationand reduced GSK-3 kinase activity by ~800% and ~25%,respectively. LY-294002 (100 µM) and wortmannin (150 µM), specificinhibitors of phosphatidylinositol 3'-kinase, attenuated IGF-I-inducedGSK-3 phosphorylation by 67 and 92%, respectively. IGF-I suppressedthe kinase activity of GSK-3. IGF-I (250 ng/ml), but not LiCl (10 mM), induced an increase in NFAT-activated luciferase reporteractivity. Cotransfection of a constitutively active GSK-3(cGSK-3) inhibited the induction by IGF-I of NFAT-inducible reporteractivity. LiCl, which inhibits GSK-3, removed the block by cGSK-3on IGF-I-inducible NFAT-responsive reporter gene activity. These datasuggest that the IGF-I-induced increase in skeletal myotube size issignaled, in part, through the inhibition of GSK-3.

     

Modulation of human neuronal alpha 1E-type calcium channel by alpha 2delta -subunit

Calcium channels are composed of a pore-forming subunit,₁, and at least two auxiliarysubunits, - and₂-subunits. It is well knownthat -subunits regulate most of the properties of the channel. Thefunction of ₂-subunit isless understood. In this study, the effects of the calcium channel₂-subunit on the neuronal_1E voltage-gated calciumchannel expressed in Xenopus oocyteswas investigated without and with simultaneous coexpression of eitherthe _1b- or the_2a-subunit. Most aspects of_1E function were affected by₂. Thus₂ caused a shift in thecurrent-voltage and conductance-voltage curves toward more positivepotentials and accelerated activation, deactivation, and theinstallation of the inactivation process. In addition, the efficiencywith which charge movement is coupled to pore opening assessed bydetermining ratios of limiting conductance to limiting charge movementwas decreased by ₂ byfactors that ranged from 1.6 (P < 0.01) for _1E-channels to 3.0 (P < 0.005) for_1E1b-channels. These results indicate that₂ facilitates the expressionand the maturation of_1E-channels and converts thesechannels into molecules responding more rapidly to voltage.

     

p38 MAPK mediates renal tubular cell TNF-{alpha} production and TNF-{alpha}-dependent apoptosis during simulated ischemia

Ischemia causes renal tubular cellloss through apoptosis; however, the mechanisms of this processremain unclear. Using the renal tubular epithelial cell lineLLC-PK₁, we developed a model of simulated ischemia(SI) to investigate the role of p38 MAPK (mitogen-activated proteinkinase) in renal cell tumor necrosis factor- (TNF-) mRNAproduction, protein bioactivity, and apoptosis. Resultsdemonstrate that 60 min of SI induced maximal TNF- mRNA productionand bioactivity. Furthermore, 60 min of ischemia induced renaltubular cell apoptosis at all substrate replacement time pointsexamined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF- mRNA production andTNF- bioactivity, and both p38 MAPK inhibition and TNF- neutralization (anti-porcine TNF- antibody) preventedapoptosis after 60 min of SI. These results constitute theinitial demonstration that 1) renal tubular cells produceTNF- mRNA and biologically active TNF- and undergoapoptosis in response to SI, and 2) p38 MAPKmediates renal tubular cell TNF- production and TNF--dependent apoptosis after SI.