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-.

     

Transforming growth factor-beta 1 modulates the expression of vascular endothelial growth factor by osteoblasts

Angiogenesis is essential to both normal and pathological bonephysiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-1 (TGF-1) modulates bone differentiation, matrixformation, and cytokine expression. The purpose of this study was toinvestigate the relationship between TGF-1 and VEGF expression inosteoblasts and osteoblast-like cells. Northern blot analysis revealedan early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cellsand MC3T3-E1 osteoblast-like cells after stimulation with TGF-1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increasedafter TGF-1 treatment. Actinomycin D inhibited the TGF-1-inducedpeak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-1signal transduction via a dominant-negative receptor II adenovirussignificantly decreased TGF-1 induction of VEGF mRNA. Additionally,TGF-1 induced a dose-dependent increase in VEGF protein expressionby MC3T3-E1 cells (P < 0.01).Dexamethasone similarly inhibited VEGF protein expression. BothTGF-1 mRNA and VEGF mRNA were concurrently present in rat membranousbone, and both followed similar patterns of expression during ratmandibular fracture healing (mRNA and protein). In summary,TGF-1-induced VEGF expression by osteoblasts and osteoblast-likecells is a dose-dependent event that may be intimately related to bonedevelopment and fracture healing.

     

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.

     

Osmotic pressure regulates alpha beta gamma -rENaC expressed in Xenopus oocytes

The hypothesisthat amiloride-sensitive Na⁺channels (ENaC) are involved in cell volume regulation was tested.Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used tosuperfuse Xenopus oocytes expressing-rat ENaC (-rENaC). Whole cell currents werereversibly dependent on external osmolarity. Under conditions ofswelling (70 mosM) or shrinkage (450 mosM), current amplitude decreasedand increased, respectively. In contrast, there was no change incurrent amplitude of H₂O-injectedoocytes to the above osmotic insults. Currents recorded from-rENaC-injected oocytes were not sensitive to externalCl concentration or to theK⁺ channel inhibitorBaCl₂. They were sensitive toamiloride. The concentration of amiloride necessary to inhibit one-halfof the maximal rENaC current expressed in oocytes(K_i; apparentdissociation constant) decreased in swollen cells and increased inshrunken oocytes. The osmotic pressure-inducedNa⁺ currents showed propertiessimilar to those of stretch-activated channels, including inhibition byGd³⁺ andLa³⁺, and decreased selectivityfor Na⁺.-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the firstdemonstration that -rENaC heterologously expressed inXenopus oocytes may contribute tooocyte volume regulation following shrinkage.

     

TGF-alpha reduces bradykinin-stimulated ion transport and prostaglandin release in human colonic epithelial cells

The effect of chronic exposure to transforming growth factor-(TGF-) on bradykinin-stimulated acute prostanoid production and ionsecretion in monolayers of HCA-7 colony 29 colonic epithelial cells hasbeen studied. Monolayers synthesized prostaglandinE₂ (PGE₂) at a basal rate of 2.10 ± 0.31 pg · monolayer¹ · min¹over 24 h. Bradykinin(10⁸-10⁵M) dose dependently increased acutePGE₂ release by three orders ofmagnitude. This was associated with a rise in cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC).When monolayers were primed by a 24-h exposure to TGF-, basalPGE₂ release rose to 6.31 ± 0.38 pg · monolayer¹ · min¹(TGF- concn 10 ng/ml; P = 0.001).However, the stimulation of acute prostaglandin release, intracellularcAMP, and increased SCC by bradykinin was significantly reduced bypreincubation with TGF-. Priming withPGE₂(10⁸-10⁶M) over 24 h mimicked the effect of TGF- on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF- maydownregulate acute responses to bradykinin. In vivo, TGF- could havean important modulatory function in regulating secretion underinflammatory conditions.     

Colonic H-K-ATPase alpha- and beta-subunits express ouabain-insensitive H-K-ATPase

Active K absorption in the rat distal colon is energizedby an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase -subunit (HKc) has been cloned and characterized (together with the -subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive⁸⁶Rb uptake. In contrast, HKc, when expressed in Sf9cells without a -subunit, yielded evidence of ouabain-insensitiveH-K-ATPase. Because a -subunit (HKc) has recently been clonedfrom rat colon, this present study was initiated to determine whetherH-K-ATPase and its sensitivity to ouabain are expressed when these twosubunits (HKc and HKc) are transfected into a mammalian cellexpression system. Transfection of HEK-293 cells with HKc and HKccDNAs resulted in the expression of HKc and HKc proteins andtheir delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was1) saturable as a function of increasing K concentration with aK_m for K of 0.63 mM; 2) inhibited byorthovanadate; and 3) insensitive to both ouabain andSch-28080. In parallel transfection studies with HKc and Na-K-ATPase1 cDNAs and with HKc cDNA alone, there was expression ofouabain-insensitive H-K-ATPase activity that was 60% and 21% of thatin HKc/HKc cDNA transfected cells, respectively. Ouabain-insensitive ⁸⁶Rb uptake was also identified incells transfected with HKc and HKc cDNAs. These studies establishthat HKc cDNA with HKc cDNA express ouabain-insensitiveH-K-ATPase similar to that identified in rat distal colon.

     

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.

     

Unusual degradation of alpha -beta complexes in Xenopus oocytes by beta -subunits of Xenopus gastric H-K-ATPase

The catalytic -subunit of oligomeric P-type ATPases such asNa-K-ATPase and H-K-ATPase requires association with a -subunit after synthesis in the endoplasmic reticulum (ER) to become stably expressed and functionally active. In this study, we have expressed the-subunit of Xenopus gastricH-K-ATPase (HK) in Xenopus oocytes together with -subunits of H-K-ATPase (HK) or Na-K-ATPase (NK) and have followed the biosynthesis, assembly, and cell surface expression of functional pumps. Immunoprecipitations ofXenopus HK from metabolicallylabeled oocytes show that it is well expressed and, when synthesizedwithout -subunits, can leave the ER and become fully glycosylated.Xenopus HK can associate with both coexpressed HK and NK, but the - complexes formed aredegraded rapidly in or close to the ER and do not produce functionalpumps at the cell surface as assessed by⁸⁶Rb uptake. A possibleexplanation of these results is thatXenopus HK may contain atissue-specific signal that is important in the formation or correcttargeting of functional - complexes in the stomach but thatcannot be recognized in Xenopusoocytes and in consequence leads to cellular degradation of the -complexes in this experimental system.

     

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.

     

beta-adrenergic receptor/cAMP-mediated signaling and apoptosis of S49 lymphoma cells

-Adrenergic receptor (AR) activationand/or increases in cAMP regulate growth and proliferation of a varietyof cells and, in some cells, promote cell death. In the current studieswe addressed the mechanism of this growth reduction by examiningAR-mediated effects in the murine T-lymphoma cell line S49.Wild-type S49 cells, derived from immature thymocytes(CD4⁺/CD8⁺) undergo growth arrest andsubsequent death when treated with agents that increase cAMP levels(e.g., AR agonists, 8-bromo-cAMP, cholera toxin, forskolin).Morphological and biochemical criteria indicate that this cell death isa result of apoptosis. In cyc and kin S49cells, which lack G_s and functional protein kinase A(PKA), respectively, AR activation of G_s and cAMPaction via PKA are critical steps in this apoptotic pathway. S49 cellsthat overexpress Bcl-2 are resistant to cAMP-induced apoptosis. Weconclude that AR activation induces apoptosis in immature Tlymphocytes via G_s and PKA, while overexpression ofBcl-2 prevents cell death. AR/cAMP/PKA-mediated apoptosis mayprovide a means to control proliferation of immature T cells in vivo.

     

Colonic H-K-ATPase beta -subunit: identification in apical membranes and regulation by dietary K depletion

P-type ATPasesrequire both - and -subunits for functionalactivity. Although an -subunit for colonic apical membraneH-K-ATPase (HKc) has been identified and studied, its -subunithas not been identified. We cloned putative -subunit rat colonicH-K-ATPase (HKc) cDNA that encodes a 279-amino-acid protein with asingle transmembrane domain and sequence homology to other rat-subunits. Northern blot analysis demonstrates that this HKc isexpressed in several rat tissues, including distal and proximal colon,and is highly expressed in testis and lung. HKc mRNA abundance is upregulated threefold compared with normal in distal colon but notproximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase ₁ mRNA abundance isunaltered in distal colon of K-depleted rats. Na depletion, which alsostimulates active K absorption in distal colon, does not increaseHKc mRNA abundance. Western blot analyses using a polyclonalantibody raised to a glutathioneS-transferase-HKc fusion proteinestablished expression of a 45-kDa HKc protein in both apical andbasolateral membranes of rat distal colon, but K depletion increasedHKc protein expression only in apical membranes. Physicalassociation between HKc and HKc proteins was demonstrated byWestern blot analysis performed with HKc antibody onimmunoprecipitate of apical membranes of rat distal colon and HKcantibody. Tissue-specific upregulation of this -subunit mRNA inresponse to K depletion, localization of its protein, its upregulationby K depletion in apical membranes of distal colon, and its physicalassociation with HKc protein provide compelling evidence that HKcis the putative -subunit of colonic H-K-ATPase.     

Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel

Protons regulateelectrogenic sodium absorption in a variety of epithelia, including thecortical collecting duct, frog skin, and urinary bladder. Recently,three subunits (, , ) coding for the epithelial sodium channel(ENaC) were cloned. However, it is not known whether pH regulatesNa⁺ channels directly byinteracting with one of the three ENaC subunits or indirectly byinteracting with a regulatory protein. As a first step to identifyingthe molecular mechanisms of proton-mediated regulation of apicalmembrane Na⁺ permeability inepithelia, we examined the effect of pH on the biophysical propertiesof ENaC. To this end, we expressed various combinations of -, -,and -subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clampand patch-clamp techniques. In addition, the effect of pH on the-ENaC subunit was examined in planar lipid bilayers. We report that ,,-ENaC currents were regulated by changes in intracellular pH(pH_i) but not by changes inextracellular pH (pH_o).Acidification reduced and alkalization increased channel activity by avoltage-independent mechanism. Moreover, a reduction ofpH_i reduced single-channel openprobability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited ,-ENaC, ,-ENaC, and -ENaC currents. We conclude thatpH_i but notpH_o regulates ENaC and that the-ENaC subunit is regulated directly bypH_i.     

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.

     

Intracellular signaling mechanisms of interleukin-1beta in synovial fibroblasts

The possibility that membrane depolarization of synovialfibroblasts caused by interleukin-1 (IL-1) was mediated byprotein kinase C (PKC) and Ca²⁺influx was studied using inhibitor and activator analysis. The effectof IL-1 was blocked by bisindolylmaleimide I, an inhibitor of PKC,and by the Ca²⁺ channel blockersnifedipine and verapamil. In other experiments, PKC was activated usingphorbol 12-myristate 13-acetate, andCa²⁺ influx was increased by meansof a Ca²⁺ ionophore. Simultaneousapplication of phorbol ester andCa²⁺ ionophore in the absence ofIL-1 mimicked the depolarization caused by IL-1. The results wereconsistent with the hypothesis that, under the conditions studied,activation of PKC and Ca²⁺ influxare necessary and sufficient processes in the transduction of IL-1by synovial cells leading to membrane depolarization. Theessential role of protein phosphorylation andCa²⁺ influx in the earlyelectrophysiological response of synovial fibroblasts to IL-1 wastherefore established. The role of IL-1-induced depolarization inregulating protein expression by the cells remains to be determined,but the results reported here, taken together with observations thatprotein phosphorylation and Ca²⁺influx also mediate the effect of IL-1 on protease production (1, 2), suggest that electrophysiological changes are actually part of thepathway for expression of proteases in response to IL-1.

     

Carbachol-induced desensitization of PLC-beta pathway in rat myometrium: downregulation of Gqalpha /G11alpha

In the estrogen-treated rat myometrium, carbachol increased thegeneration of inositol phosphates by stimulating the muscarinic receptor-G_q/G₁₁-phospholipaseC-3 (PLC-3) cascade. Exposure to carbachol resulted in a rapidand specific (homologous) attenuation of the subsequent muscarinicresponses in terms of inositol phosphate production, PLC-3translocation to membrane, and contraction. Refractoriness wasaccompanied by a reduction of membrane muscarinic binding sites and anuncoupled state of residual receptors. Protein kinase C (PKC) alteredthe functionality of muscarinic receptors and contributed to theinitial period of desensitization. A delayed phase of the muscarinicrefractoriness was PKC independent and was associated with adownregulation ofG_q/G₁₁.Atropine failed to induce desensitization as well asG_q/G₁₁downregulation, indicating that both events involve active occupancy ofthe receptor. Prolonged exposure toAlF₄ reduced subsequent AlF₄ as well as carbachol-mediatedinositol phosphate responses and similarly induced downregulation ofG_q/G₁₁. Data suggest that a decrease in the level ofG_q/G₁₁is subsequent to its activation and may account forheterologous desensitization.

     

Interaction of alpha- and beta-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase beta-subunit

The assembly of the -subunit of thegastric H-K-ATPase (HK) with the -subunit of the H-K-ATPase orthe Na-K-ATPase (NaK) was characterized with two anti-HKmonoclonal antibodies (MAbs). In fixed gastric oxyntic cells, inH-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cellstransfected with HK, MAb 2/2E6 was observed to bind to HK onlywhen interactions between - and -subunits were disrupted byvarious denaturants. The epitope for MAb 2/2E6 was mapped to thetetrapeptide S₂₂₆LHY₂₂₉ of the extracellulardomain of HK. The epitope for MAb 2G11 was mapped to the eightNH₂-terminal amino acids of the cytoplasmic domain ofHK. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HK with -subunits of the endogenous cell surface NaK, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HK. In HK-transfected LLC-PK₁ cells,significant immunofluorescent labeling of HK at the cell surfacecould be detected without postfixation denaturation or in live cells,although a fraction of transfected HK could also becoimmunoprecipitated with NaK. Thus assembly of HK with NaKdoes not appear to be a stringent requirement for cell surface deliveryof HK in LLC-PK₁ cells but may be required in MDCKcells. In addition, endogenous posttranslational regulatory mechanismsto prevent hybrid - heterodimer assembly appear to be compromisedin transfected cultured renal epithelial cells. Finally, theextracellular epitope for assembly-sensitive MAb 2/2E6 may represent aregion of HK that is associated with - interaction.

     

Hyaluronidase enhancement of TNF-mediated cell death is reversed by TGF-beta 1

Bothhyaluronidase and transforming growth factor (TGF)-1 play asignificant role in the development of prostate cancer. In this study,the regulation of tumor necrosis factor (TNF)-mediated cell death byhyaluronidase and TGF-1 was investigated. Preexposure of L929fibroblasts, prostate LNCaP cells, and epithelial Mv 1 Lu cells tohyaluronidase for a minimum of 12 h resulted in significant enhancementof cell death by TNF. Phosphorylation of p42 and p44 mitogen-activatedprotein (MAP) kinases was found by stimulation of L929 cells withhyaluronidase for 30 min, indicating that the Raf/MAPkinase-extracellular signal-regulating protein kinase (MEK)/MAP kinase pathway was activated. However, blocking the activation of upstream MAP kinase kinase (MEK 1 and 2 kinase) byPD-98059 failed to inhibit the hyaluronidase-enhanced TNF killing ofcells, suggesting that hyaluronidase-mediated degradation of extracellular matrix and membrane components may elicit multiple signaling pathways. As a potent stimulator of extracellular matrix protein synthesis, TGF-1 blocked the hyaluronidase-enhanced death ofL929 and LNCaP cells mediated by TNF. TGF-1 activatedprotein-tyrosine kinases in L929 cells, in which the tyrosine kinaseinhibitors lavendustin A and tyrphostin blocked the activation as wellas the TGF-1 inhibition of hyaluronidase effects. Functionalantagonism was also observed between hyaluronidase and TGF-1 in cellgrowth regulation. For example, TGF-1-mediated suppression ofepithelial Mv 1 Lu cell growth was abolished by hyaluronidase. Overall,it is demonstrated in this study that hyaluronidase reciprocally antagonized TGF-1 in the modulation of cell proliferation and TNF-mediated death.

     

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.

     

Acute glucose-induced downregulation of PKC-betaII accelerates cultured VSMC proliferation

Accelerated vascular smooth muscle cell(VSMC) proliferation contributes to the formation of atheroscleroticlesions. To investigate protein kinase C (PKC)-II functions withregard to glucose-induced VSMC proliferation, human VSMC from aorta(AoSMC), a clonal VSMC line of rat aorta (A10), and A10 cellsoverexpressing PKC-I (I-A10) and PKC-II (II-A10) werestudied with the use of three techniques to evaluate glucose effects onaspects affecting proliferation. High glucose (25 mM) increased DNAsynthesis and accelerated cell proliferation compared with normalglucose (5.5 mM) in AoSMC and A10 cells, but not in I-A10 andII-A10 cells. The PKC-II specific inhibitor CGP-53353 inhibitedglucose-induced cell proliferation and DNA synthesis in AoSMC and A10cells. In flow cytometry analysis, high glucose increased thepercentage of A10 cells at 12 h after cell cycle initiation butdid not increase the percentage of I-A10 or II-A10 cells enteringS phase. PKC-II protein levels decreased before the peak of DNAsynthesis, and high glucose further decreased PKC-II mRNA andprotein levels in AoSMC and A10 cells. These results suggest that highglucose downregulates endogenous PKC-II, which then alters thenormal inhibitory role of PKC-II in cell cycle progression,resulting in the stimulation of VSMC proliferation through acceleration of the cell cycle.

     

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2)

This study examined the ability of protein kinase C (PKC) toinduce heterologous desensitization by targeting specific G proteinsand limiting their ability to transduce signals in smooth muscle.Activation of PKC by pretreatment of intestinal smooth muscle cellswith phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, orthe phosphatase 1 and phosphatase 2A inhibitor, calyculin A,selectively phosphorylated G_i-1 and G_i-2,but not G_i-3 or G_o, and blockedinhibition of adenylyl cyclase mediated by somatostatin receptorscoupled to G_i-1 and opioid receptors coupled toG_i-2, but not by muscarinic M₂ and adenosineA₁ receptors coupled to G_i-3. Phosphorylationof G_i-1 and G_i-2 and blockade of cyclaseinhibition were reversed by calphostin C and bisindolylmaleimide, andadditively by selective inhibitors of PKC and PKC. Blockade ofinhibition was prevented by downregulation of PKC. Phosphorylation ofG-subunits by PKC also affected responses mediated by-subunits. Pretreatment of muscle cells withcANP-(4-23), a selective agonist of the natriureticpeptide clearance receptor, NPR-C, which activates phospholipase C(PLC)-3 via the -subunits of G_i-1 andG_i-2, inhibited the PLC- response to somatostatin and[D-Pen^2,5]enkephalin. The inhibition waspartly reversed by calphostin C. Short-term activation of PKC had noeffect on receptor binding or effector enzyme (adenylyl cyclase orPLC-) activity. We conclude that selective phosphorylation ofG_i-1 and G_i-2 by PKC partly accounts forheterologous desensitization of responses mediated by the - and-subunits of both G proteins. The desensitization reflects adecrease in reassociation and thus availability of heterotrimeric G proteins.