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.

     

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.

     

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.

     

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.

     

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.

     

Differential requirement for NF-kappaB-inducing kinase in the induction of NF-kappaB by IL-1beta,TNF-alpha,and Fas

In this study, weexamined the role of the nuclear factor-B (NF-B)-inducing kinase(NIK) in distinct signaling pathways leading to NF-B activation. Weshow that a dominant-negative form of NIK (dnNIK) delivered byadenoviral (Ad5dnNIK) vector inhibits Fas-induced IBphosphorylation and NF-B-dependent gene expression in HT-29 and HeLacells. Interleukin (IL)-1- and tumor necrosis factor-(TNF-)-induced NF-B activation and B-dependent gene expressionare inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells.Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF--inducedapoptosis at an early time point. However, cytokine- andFas-induced signals to NF-B are finally integrated by the IBkinase (IKK) complex, since IB phosphorylation, NF-B DNAbinding activity, and IL-8 gene expression were strongly inhibited inHT-29 and HeLa cells overexpressing dominant-negative IKK(Ad5dnIKK). Our findings support the concept that cytokine signalingto NF-B is redundant at the level of NIK. In addition, this studydemonstrates for the first time the critical role of NIK and IKK inFas-induced NF-B signaling cascade.

     

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-.

     

Effect of interleukin-1beta and tumor necrosis factor-alpha on gene expression in human endothelial cells

Interleukin-1(IL-1) and tumor necrosis factor- (TNF-) are two majorcytokines that rise to relatively high levels during systemicinflammation, and the endothelial cell (EC) response to these cytokinesmay explain some of the dysfunction that occurs. To better understandthe cytokine-induced responses of EC at the gene expression level,human umbilical vein EC were exposed to IL-1 or TNF- for varioustimes and subjected to cDNA microarray analyses to study alterations intheir mRNA expression. Of ~4,000 genes on the microarray, expressionlevels of 33 and 58 genes appeared to be affected by treatment withIL-1 and TNF-, respectively; 25 of these genes responded to bothtreatments. These results suggest that the effects of IL-1 andTNF- on EC are redundant and that it may be necessary to suppressboth cytokines simultaneously to ameliorate the systemic response.

     

Protein kinase Calpha participates in activation of store-operated Ca2+ channels in human glomerular mesangial cells

Protein kinase C (PKC) plays animportant role in activating store-operated Ca²⁺ channels(SOC) in human mesangial cells (MC). The present study was performed todetermine the specific isoform(s) of conventional PKC involved inactivating SOC in MC. Fura 2 fluorescence ratiometry showed that thethapsigargin-induced Ca²⁺ entry (equivalent to SOC) wassignificantly inhibited by 1 µM Gö-6976 (a specific PKC andI inhibitor) and PKC antisense treatment (2.5 nM for 24-48h). However, LY-379196 (PKC inhibitor) and2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanoldimethyl ether(HBDDE; PKC and  inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single-channel analysis in the cell-attached configuration revealed that Gö-6976 and PKC antisensesignificantly depressed thapsigargin-induced activation of SOC.However, LY-379196 and HBDDE did not affect the SOC responses. Ininside-out patches, application of purified PKC or I, but notII or , significantly rescued SOC from postexcision rundown.Western blot analysis revealed that thapsigargin evoked a decrease incytosolic expression with a corresponding increase in membraneexpression of PKC and . However, the translocation from cytosolto membranes was not detected for PKCI or II. These resultssuggest that PKC participates in the intracellular signaling pathwayfor activating SOC upon release of intracellular stores ofCa²⁺.

     

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.

     

Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages

The APO-1/Fasligand (FasL) and tumor necrosis factor- (TNF-) are twofunctionally related molecules that induce apoptosis ofsusceptible cells. Although the two molecules have been reported toinduce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-, raising thepossibility that TNF- may be involved in FasL-inducedapoptosis. Because TNF- gene expression is under the controlof nuclear factor-B (NF-B), we investigated whether FasL caninduce NF-B activation and whether such activation plays a role inFasL-mediated cell death in macrophages. Gene transfection studiesusing NF-B-dependent reporter plasmid showed that FasL did activateNF-B promoter activity. Gel shift studies also revealed that FasLmobilized the p50/p65 heterodimeric form of NF-B. Inhibition ofNF-B by a specific NF-B inhibitor, caffeic acid phenylethylester, or by dominant expression of the NF-B inhibitory subunitIB caused an increase in FasL-induced apoptosis and areduction in TNF- expression. However, neutralization of TNF- byspecific anti-TNF- antibody had no effect on FasL-inducedapoptosis. These results indicate that FasL-mediated cell deathin macrophages is regulated through NF-B and is independent ofTNF- activation, suggesting the antiapoptotic role of NF-Band a separate death signaling pathway mediated by FasL.

     

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.

     

TGF-beta effects on epithelial ion transport and barrier: reduced Cl- secretion blocked by a p38 MAPK inhibitor

Growthfactors affect a variety of epithelial functions. We examined theability of TGF- to modulate epithelial ion transport andpermeability. Filter-grown monolayers of human colonic epithelia, T84and HT-29 cells, were treated with TGF- (0.1-100 ng/ml,15 min-72 h) or infected with an adenoviral vector encodingTGF- (Ad-TGF) for 144 h. Ion transport (i.e., short-circuitcurrent, I_sc) and transepithelial resistance(TER) were assessed in Ussing chambers. Neither recombinant TGF- norAd-TGF infection affected baseline I_sc;however, exposure to 1 ng/ml TGF- led to a significant (30-50%) reduction in the I_sc responses toforskolin, vasoactive intestinal peptide, and cholera toxin (agentsthat evoke Cl secretion via cAMP mobilization) and to thecell-permeant dibutyryl cAMP. Pharmacological analysis of signalingpathways revealed that the inhibition of cAMP-driven epithelialCl secretion by TGF- was blocked by pretreatment withSB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors ofJNK, ERK1/2 MAPK, or phosphatidylinositol 3'-kinase. TGF- enhanced the barrier function of the treated monolayers by up to threefold asassessed by TER; however, this event was temporally displaced from thealtered I_sc response, being statisticallysignificant only at 72 h posttreatment. Thus, in addition toTGF- promotion of epithelial barrier function, we show that thisgrowth factor also reduces responsiveness to cAMP-dependentsecretagogues in a chronic manner and speculate that this serves as abraking mechanism to limit secretory enteropathies.

     

IkappaBalpha-dependent regulation of low-shear flow-induced NF-kappa B activity: role of nitric oxide

We have investigated the role ofinhibitor B (IB) in the activation of nuclear factor B(NF-B) observed in human aortic endothelial cells (HAEC) undergoinga low shear stress of 2 dynes/cm². Low shear for 6 hresulted in a reduction of IB levels, an activation of NF-B,and an increase in B-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion.Overexpression of IB in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation ofIB is the major factor in the low shear-induced activation ofNF-B in HAEC. We then investigated the role of nitric oxide (NO) inthe regulation of IB/NF-B. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-B activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 µM) orsodium nitroprusside (1 mM) before low shear stress significantlyincreased cytoplasmic IB and concomitantly reduced NF-Bbinding activity and B-dependent VCAM-1 promoter activity. Together,these data suggest that NO may play a major role in the regulation ofIB levels in HAEC and that the application of low shear flowincreases NF-B activity by attenuating NO generation and thusIB levels.

     

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.

     

Vascular endothelial cells express isoforms of protein kinase A inhibitor

First published September 5, 2001;10.1152/ ajpcell.00256.2001.The expression and function of theendogenous inhibitor of cAMP-dependent protein kinase (PKI) inendothelial cells are unknown. In this study, overexpression of rabbitmuscle PKI gene into endothelial cells inhibited the cAMP-mediatedincrease and exacerbated thrombin-induced decrease in endothelialbarrier function. We investigated PKI expression in human pulmonaryartery (HPAECs), foreskin microvessel (HMECs), and brain microvesselendothelial cells (HBMECs). RT-PCR using specific primers for humanPKI, human PKI, and mouse PKI sequences detectedPKI and PKI mRNA in all three cell types. Sequencing and BLASTanalysis indicated that forward and reverse DNA strands for PKI andPKI were of >96% identity with database sequences. RNaseprotection assays showed protection of the 542 nucleotides in HBMEC andHPAEC PKI mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKImRNA. Western blot analysis indicated that PKI protein was detectedin all three cell types, whereas PKI was found in HBMECs. Insummary, endothelial cells from three different vascular beds expressPKI and PKI, which may be physiologically important inendothelial barrier function.

     

Combined modulation of the mesangial machinery for monocyte recruitment by inhibition of NF-kappa B

The activation of nuclear factor-B(NF-B) is required for the induction of many of the adhesionmolecules and chemokines involved in the inflammatory leukocyterecruitment to the kidney. Here we studied the effects of NF-Binhibition on the machinery crucial for monocyte infiltration of theglomerulus during inflammation. In mesangial cells (MC), the proteaseinhibitors MG-132 and N--tosyl-L-lysine chloromethyl ketone or adenoviral overexpression of IB- prevented the complete IB- degradation following tumor necrosis factor- (TNF-) stimulation. This resulted in a marked inhibition ofTNF--induced expression of mRNA and protein for the immunoglobulinmolecules intracellular adhesion molecule-1 and vascular cell adhesionmolecule-1 and the chemokines growth-related oncogene-, monocytechemoattractant protein-1, interleukin-8, or fractalkine in MC.Finally, the inhibition of IB- degradation or IB-overexpression suppressed the chemokine-induced transendothelialmonocyte chemotaxis toward MC and the chemokine-triggered firm adhesionof monocytic cells to MC. The inhibition of NF-B by pharmacologicalintervention or gene transfer may present a multimodal approach tocontrol the machinery propagating inflammatory recruitment of monocytesduring glomerular disease.

     

Decreased 5alpha-dihydrotestosterone catabolism suppresses T lymphocyte functions in males after trauma-hemorrhage

Trauma-hemorrhage producesprofound immunosuppression in males but not in proestrus females.Prior castration or flutamide treatment of males followingtrauma-hemorrhage prevents immunosuppression, implicating5-dihydrotestosterone for the immunosuppressive effects. 5-Dihydrotestosterone, a high-affinity androgen receptor-binding steroid, is synthesized in tissues as needed and seldom accumulates. The presence of steroidogenic enzymes in T lymphocytes suggests bothsynthesis and catabolism of 5-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5-dihydrotestosterone activity inT lymphocytes of males following trauma-hemorrhage is due to decreasedcatabolism. Accordingly, catabolism of 5-dihydrotestosterone wasassessed in splenic T lymphocytes by examining the activity andexpression of enzymes involved. Analysis showed increased synthesis anddecreased catabolism of 5-dihydrotestosterone in intact male Tlymphocytes following trauma-hemorrhage. In contrast, reduced5-reductase activity and increased expression of17-hydroxysteroid dehydrogenase oxidative isomers suggestinactivation of 5-dihydrotestosterone in precastrated males. Thusour study suggests increased synthesis and decreased catabolism of5-dihydrotestosterone as a reason for loss of T lymphocyte functionsin intact males following trauma-hemorrhage, as evidenced by decreasedrelease of interleukin-2 and -6.

     

Functional overload increases beta-MHC promoter activity in rodent fast muscle via the proximal MCAT (betae3) site

Functional overload (OL)of the rat plantaris muscle by the removal of synergistic musclesinduces a shift in the myosin heavy chain (MHC) isoform expressionprofile from the fast isoforms toward the slow type I, or, -MHCisoform. Different length rat -MHC promoters were linked to afirefly luciferase reporter gene and injected in control and OLplantaris muscles. Reporter activities of 3,500, 914, 408, and215 bp promoters increased in response to 1 wk of OL. The smallest171 bp promoter was not responsive to OL. Mutation analyses ofputative regulatory elements within the 171 and 408 bp region wereperformed. The 408 bp promoters containing mutations of the e1,distal muscle CAT (MCAT; e2), CACC, or A/T-rich (GATA), were stillresponsive to OL. Only the proximal MCAT (e3) mutation abolished theOL response. Gel mobility shift assays revealed a significantly higherlevel of complex formation of the e3 probe with nuclear protein fromOL plantaris compared with control plantaris. These results suggestthat the e3 site functions as a putative OL-responsive element inthe rat -MHC gene promoter.