Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E₂) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E₂ pellets were removed, but administration of progesterone (P), 5-dihydrotestosterone (DHT), or continued implantation of E₂ pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E₂, P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.     

Biotransformation of paeonol by Panax ginseng root and cell cultures

Panax ginseng root and cell cultures were shown to biotransform paeonol (1) into its 2-O-β-d-glucopyranoside (2). P. ginseng root cultures were also able to biotransform paeonol (1) into its 2-O-β-d-xylopyranoside (3), 2-O-β-d-glucopyranosyl(1 → 6)-β-d-glucopyranoside (4) and 2-O-β-d-xylopyranosyl(1 → 6)-β-d-glucopyranoside (5), and its demethylated derivate, 2′,4′-dihydroxyacetophenone (6). Compounds 3 and 4 are new glycosides. It is the first example that the administrated compound was converted into its xylopyranoside by plant biotransformation.     

An acetophenone and three naphthalides from turkish Rhamnus libanoticus

A new acetophenone glycoside and two new naphthalide glycosides have been isolated from the bark of Turkish Rhamnus libanoticus together with 7-hydroxy-5-methoxyphthalide 7-O-β-D-glucoside. The structures of the new compounds were elucidated by spectroscopic methods as 2,6-dihydroxy-4-methoxyacetophenone 2-O-β- rutinoside, 8,9-dihydroxy-6-methoxynaphthalide 8-O-β-rutinoside, 8,9-dihydroxy-6-methoxynaphthalide 8-O-/3b-D glucoside, respectively.     

Streptococcus pneumoniae type XIV polysaccharide: synthesis of a repeating branched tetrasaccharide with dioxa-type spacer-arms

β-Glycosides of 2-acetamido-2-deoxy- -glucopyranose were synthesized, using either 7-methoxycarbonyl-3,6-dioxa-1-heptanol or 8-azido-3,6-dioxa-1-octanol. Selective β-lactosylation of 7-methoxycarbonyl-3,6-dioxaheptyl 2-acetamido-3-O-benzyl-2-deoxy-β- -glucopyranoside with hepta-O-acetyl-lactosyl-trichloroacetimidate, followed by β-galactosylation of the secondary hydroxyl group with O-(2,3,4,6-tetra-O-acetyl-- -galactopyranosyl)trichloroacetimidate, catalytic hydrogenolysis, and O-deacetylation, gave 7-methoxycarbonyl-3,6-dioxaheptyl 2-acetamido-2-deoxy-4-O-β- -galactopyranosyl-6-O-(4-O-β- -galactopyranosyl-β- -glucopyranosyl)β- -glucopyranoside. Selective β-lactosylation of 8-azido-3,6-dioxaocytl 2-acetamido-3-O-benzyl-2-deoxy-β- -glucopyranoside with hepta-O-acetyl-lactosyl bromide in the presence of silver triflate, followed by condensation with 2,3,4,6-tetra-O-acetyl-- -galactopyranosyl bromide in the presence of silver triflate, catalytic hdyrogenolysis, and O-deacetylation, gave 8-azido-3,6-dioxaoctyl 2-acetamido-2-deoxy-4-O-β- -galactopyranosyl-6-O-(4-O-β- -galactopyranosyl-β- -glucopyranosyl)-β- glucopyranoside.     

Cycloartane triterpene glycosides from Astragalus trigonus

Three new cycloartane glycosides, trigonoside I, II and III, and the known astragalosides I and II were isolated from the roots of Astragalus trigonus. The structures of the new glycosides were totally elucidated by high field (600 MHz) NMR analyses as cycloastragenol-6-O-β-xylopyranoside, cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-xylopyranosyl]-6-O-β- d-xylopyranoside and cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-(3-O-acetyl)-xylopyranosyl]-6-O-β-d-xylopyranoside.     

Acylated flavonol diglucosides from Lotus polyphyllos

Three acylated flavonol diglucosides, kaempferol 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; quercetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; isorhamnetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside were isolated from the whole plant aqueous alcohol extract of Lotus polyphyllos. The known 3,7-di-O-glucosides of the aglycones kaempferol, quercetin and isorhamnetin were also characterized. All structures were established on the basis of chemical and spectral evidence.     

Six new C21 steroidal glycosides from Asclepias curassavica L

Six new C₂₁ steroidal glycosides, named curassavosides A–F (3–8), were obtained from the aerial parts of Asclepias curassavica (Asclepiadaceae), along with two known oxypregnanes, 12-O-benzoyldeacylmetaplexigenin (1) and 12-O-benzoylsarcostin (2). By spectroscopic methods, the structures of the six new compounds were determined as 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (3), 12-O-benzoylsarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (4), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (5), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-digitoxopyranoside (6), 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (7), and 12-O-benzoylsarcostin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (8), respectively. All compounds (1–8) were tested for in vitro cytotoxicity; only compound 3 showed weak inhibitory activity against Raji and AGZY cell lines.     

Triterpenoidal saponins from Dumasia Truncata

Extraction of the aerial parts of Dumasia truncata Sieb et Zucc. afforded two new triterpenoidal saponins, together with four known ones. The structures of the new compounds were elucidated by spectral analysis as 3-O--l-rhamnopyranosyl-(1 → 3)-β-d-glucuronopyranosy-28-O-β-d-glucopyransoyl hederagenin and 3-O-β-d-xylopyranosyl-(1 → 2)-[-l-rhamnopyranosyl(1 → 3)]-β-d-glucuronopyranosyl oleanic acid.     

Pin1 Promotes Transforming Growth Factor-��-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.     

Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) prevents ornithine decarboxylase inhibition and apoptosis in L1210 leukemic cells exposed to TGF-β1

Previous studies have shown that growth suppression and apoptosis of leukemic cells exposed to TGF-β₁ is associated with the inhibition of ornithine decarboxylase (ODC) — the key enzyme of polyamine pathway. The aim of the present study was to evaluate the influence of 12-O-tetradecanoylphorbol 13-acetate (TPA) — a potent ODC inducer on antiproliferative and apoptotic effects of TGF-β₁ in L1210 leukemic cells. Cells were incubated in 2%FCS/RPMI1640 medium, supplemented with TGF-β₁ (2 ng/ml), TPA (100 ng/ml) or -difluoromethyl-ornithine (DFMO) (5 mM). Cell proliferation, apoptosis and necrosis were evaluated using [methyl-³H] thymidine, electron microscopy, electrophoresis of DNA and trypan blue exclusion. Expression and activity of ODC were determinated by RT-PCR and measurement of ¹⁴CO₂ release from L-1-¹⁴C ornithine, respectively. TGF-β₁ inhibited proliferation and induced apoptotic and necrotic cell death in L1210 leukemic cells. The above effects were associated with the inhibition of ODC expression and activity, measured 2 and 4 hr after TGF-β₁ administration, respectively. The presence of DFMO, an irreversible inhibitor of ODC, led to apoptotic fragmentation of DNA, similar to that observed in TGF-β₁-treated cultures. Administration of TPA simultaneously with TGF-β₁ significantly reduced antiproliferative, apoptotic and necrotic effects of TGF-β₁, and prevented its inhibitory action on ODC expression and activity. It is concluded that: down-regulation of ODC expression may be one of the early events associated with TGF-β₁-evoked suppression of growth and apoptosis; ODC is involved in the mechanism of protective action of TPA on TGF-β₁-related growth inhibition of L1210 leukemic cells.     

Macrophage LRP1 suppresses neo-intima formation during vascular remodeling by modulating the TGF-β signaling pathway

Background

Vascular remodeling in response to alterations in blood flow has been shown to modulate the formation of neo-intima. This process results from a proliferative response of vascular smooth muscle cells and is influenced by macrophages, which potentiate the development of the intima. The LDL receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that recognizes a number of ligands including apoE-containing lipoproteins, proteases and protease-inhibitor complexes. Macrophage LRP1 is known to influence the development of atherosclerosis, but its role in vascular remodeling has not been investigated.

Methodology/Principal Findings

To define the contribution of macrophage LRP1 to vascular remodeling, we generated macrophage specific LRP1-deficient mice (macLRP1-/-) on an LDL receptor (LDLr) knock-out background. Using a carotid ligation model, we detected a 2-fold increase in neointimal thickening and a 2-fold increase in the intima/media ratio in macLRP1-/- mice. Quantitative RT-PCR arrays of the remodeled vessel wall identified increases in mRNA levels of the TGF-β2 gene as well as the Pdgfa gene in macLRP1-/- mice which could account for the alterations in vascular remodeling. Immunohistochemistry analysis revealed increased activation of the TGF-β signaling pathway in macLRP1-/- mice. Further, we observed that LRP1 binds TGF-β2 and macrophages lacking LRP1 accumulate twice as much TGF-β2 in conditioned media. Finally, TNF-α modulation of the TGF-β2 gene in macrophages is attenuated when LRP1 is expressed. Together, the data reveal that LRP1 modulates both the expression and protein levels of TGF-β2 in macrophages.

Conclusions/Significance

Our data demonstrate that macrophage LRP1 protects the vasculature by limiting remodeling events associated with flow. This appears to occur by the ability of macrophage LRP1 to reduce TGF-β2 protein levels and to attenuate expression of the TGF-β2 gene resulting in suppression of the TGF-β signaling pathway.     

Flavonol glycosides from Pteridium aquilinum

Two new flavonol glycosides from aerial parts of Pteridium aquilinum were identified as quercetin 3-O-β-laminaribioside and isorhamnetin 3-o-β-laminaribioside by chemical and spectroscopic methods.     

Rosiglitazone inhibits transforming growth factor-β1 mediated fibrogenesis in ADPKD cyst-lining epithelial cells

Background

Interstitial fibrosis plays an important role in progressive renal dysfunction in autosomal dominant polycystic kidney disease (ADPKD). In our previous studies, we confirmed that PPAR-γ agonist, rosiglitazone could protect renal function and prolong the survival of a slowly progressive ADPKD animal model by reducing renal fibrosis. However, the mechanism remains unknown.

Methods

Primary culture epithelial cells pretreated with TGF-β1 were incubated with rosiglitazone. Extracellular matrix proteins were detected using real-time PCR and Western blotting. MAPK and Smad2 phosphorylation were measured with western blot. ERK1/2 pathway and P38 pathway were inhibited with the specific inhibitors PD98059 and SB203580. The Smad2 pathway was blocked with the siRNA. To address whether PPAR-γ agonist-mediated inhibition of TGF-β1–induced collagen type I expression was mediated through a PPAR-γ dependent mechanism, genetic and pharmaceutical approaches were used to block the activity of endogenous PPARγ.

Results

TGF-β1-stimulated collagen type I and fibronectin expression of ADPKD cyst-lining epithelia were inhibited by rosiglitazone in a dosage-dependent manner. Smad2, ERK1/2 and P38 pathways were activated in response to TGF-β1; however, TGF-β1 had little effect on JNK pathway. Rosiglitazone suppressed TGF-β1 induced Smad2 activation, while ERK1/2 and P38MAPK signals remained unaffected. Rosiglitazone could also attenuate TGF-β1-stimulated collagen type I and fibronectin expression in primary renal tubular epithelial cells, but had no effect on TGF-β1–induced activation of Smad2, ERK1/2 and P38 pathways. There was no crosstalk between the Smad2 and MAPK pathways in ADPKD cyst-lining epithelial cells. These inhibitory effects of rosiglitazone were reversed by the PPARγ specific antagonist GW9662 and PPARγ siRNA.

Conclusion

ADPKD cyst-lining epithelial cells participate in TGF-β1 mediated fibrogenesis. Rosiglitazone could suppress TGF-β1–induced collagen type I and fibronectin expression in ADPKD cyst-lining epithelia through modulation of the Smad2 pathway. Our study may provide therapeutic basis for clinical applications of rosiglitazone in retarding the progression of ADPKD.     

Identification of retinoic acid in a high content screen for agents that overcome the anti-myogenic effect of TGF-beta-1

Background

Transforming growth factor beta 1 (TGF-β1) is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. When neutralizing antibodies to TGF-β1 or the angiotensin II inhibitor losartan were used to reduce TGF-β1 signaling, muscle morphology and function were restored in mouse models of Marfan Syndrome and muscular dystrophy. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-β1.

Methodology/Principal Findings

A high-content cell-based assay was developed in a 96-well plate format that detects the expression of myosin heavy chain (MHC) in C2C12 cells. The assay was used to quantify the dose-dependent responses of C2C12 cell differentiation to TGF-β1 and to the TGF-β1 Type 1 receptor kinase inhibitor, SB431542. Thirteen agents previously described as promoting C2C12 differentiation in the absence of TGF-β1 were screened in the presence of TGF-β1. Only all-trans retinoic acid and 9-cis retinoic acid allowed a maximal level of C2C12 cell differentiation in the presence of TGF-β1; the angiotensin-converting enzyme inhibitor captopril and 10 nM estrogen provided partial rescue. Vitamin D was a potent inhibitor of retinoic acid-induced myogenesis in the presence of TGF-β1. TGF-β1 inhibits myoblast differentiation through activation of Smad3; however, retinoic acid did not inhibit TGF-β1-induced activation of a Smad3-dependent reporter gene in C2C12 cells.

Conclusions/Significance

Retinoic acid alleviated the anti-myogenic effect of TGF-β1 by a Smad3-independent mechanism. With regard to the goal of improving muscle regeneration and function in individuals with muscle disease, the identification of retinoic acid is intriguing in that some retinoids are already approved for human therapy. However, retinoids also have well-described adverse effects. The quantitative, high-content assay will be useful to screen for less-toxic retinoids or combinations of agents that promote myoblast differentiation in the presence of TGF-β1.     

Effects of 9-ene-tetrahydrocannabinol on expression of β-type transforming growth factors, insulin-like growth factor-I and c-myc genes in the mouse uterus

Effects of cannabinoid on expression of β-type transforming growth factors (TGF-β1, -β2 and -β3), insulin-like growth factor-I (IGF-I) and c-myc genes in the uteri of adult ovariectomized mice were examined using Northern blot hybridization. Mice were exposed to 9-ene-tetrahydrocannabinol (THC) alone or in combination with an injection of estradiol-17β (E₂) and/or progesterone (P₄), and uteri were analyzed at various times thereafter. TGF-β isoform messenger RNAs (mRNAs) persisted in ovariectomized uteri and their levels were not altered after THC treatment, whereas an injection of E₂ caused a modest increase in TGF-β1 and -β3 mRNA levels at 24 h. Imposition of THC treatment advanced the stimulatory effects of E₂ by changing the timing for the peak of TGF-β3 mRNA levels to 12 h. In comparison, E₂ treatment substantially elevated the levels of TGF-β2 mRNA at 6 h, and THC potentiated this E₂ response without affecting the timing for the response. Imposition of P₄ treatment did not antagonize any of these responses. P₄ treatment alone or with THC had insignificant effects on mRNA levels for these TGF-β isoforms. Uterine levels of IGF-I and c-myc mRNAs were low in ovariectomized mice and THC did not alter these mRNA levels. In contrast, E₂ treatment induced a rapid, but transient, increase in IGF-I and c-myc mRNAs, and THC antagonized the rapid c-myc mRNA response and altered the timing of the IGF-I mRNA response. P₄ treatment alone also caused the transient induction of these mRNAs, but THC failed to antagonize these effects. An injection of P₄ plus E₂ resulted in further modest increases in IGF-I and c-myc mRNA levels as compared to E₂ or P₄ treatment alone. However, THC did not antagonize these transient stimulatory effects of the combined ovarian steroids. The data suggest that THC should not be classified as estrogenic or antiestrogenic. However, this compound can modulate (potentiate, antagonize and/or alter timing) the effects of ovarian steroids on uterine gene expression.     

Poly(ADP-ribose) polymerase 1 is indispensable for transforming growth factor-β Induced Smad3 activation in vascular smooth muscle cell

Background

Transforming growth factor type-β (TGF-β)/Smad pathway plays an essential role in vascular fibrosis. Reactive oxygen species (ROS) generation also mediates TGF-β signaling-induced vascular fibrosis, suggesting that some sort of interaction exists between Smad and redox pathways. However, the underlying molecular mechanism is largely unknown. This study aims to investigate the influence of poly(ADP-ribose) polymerase 1 (PARP1), a downstream effector of ROS, on TGF-β signaling transduction through Smad3 pathway in rat vascular smooth muscle cells (VSMCs).

Methods and Results

TGF-β1 treatment promoted PARP1 activation through induction of ROS generation in rat VSMCs. TGF-β1-induced phosphorylation and nuclear accumulation of Smad3 was prevented by treatment of cells with PARP inhibitor, 3-aminobenzamide (3AB) or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetami (PJ34), or PARP1 siRNA. TGF-β1 treatment promoted poly(ADP-ribosy)lation of Smad3 via activation of PARP1 in the nucleus. Poly(ADP-ribosy)lation enhanced Smad-Smad binding element (SBE) complex formation in nuclear extracts and increased DNA binding activity of Smad3. Pretreatment with 3AB, PJ34, or PARP1 siRNA prevented TGF-β1-induced Smad3 transactivation and expression of Smad3 target genes, including collagen Iα1, collagen IIIα1 and tissue inhibitor of metalloproteinase 1, in rat VSMCs.

Conclusions

PARP1 is indispensable for TGF-β1 induced Smad3 activation in rat VSMCs. Targeting PARP1 may be a promising therapeutic approach against vascular diseases induced by dysregulation of TGF-β/Smad3 pathway.     

Interactions between FSH, estradiol-17β and transforming growth factor-β regulate growth and differentiation in the rat gonad

Estradiol-17β (E₂) is a mitogen in vivo for the proliferation of granulosa cells in the rat ovary. E₂ is synthesized by the preovulatory follicle through a series of gonadotrophin-dependent events: LH stimulates thecal cells to synthesize androgens (androstenedione and testosterone) which are substrates for FSH-induced aromatization to estrogens in granulosa cells. More recently, we have found that transforming growth factor-β (TGF-β) stimulates DNA synthesis in rat granulosa cells in vitro and this effect is augmented by FSH. Since E₂ is a mitogen in vivo and TGF-β is the only known growth factor to stimulate proliferation in vitro, the possible link between the actions of E₂ and TGF-β were examined. E₂ stimulated the secretion of a TGF-β-like factor by rat granulosa cells in culture, and with time DNA synthesis was stimulated. The mitogenic action of E₂ was enhanced in the presence of FSH, and attenuated by a neutralizing antibody to TGF-β. The latter observations have identified TGF-β as the “missing-link” in the mitogenic actions of E₂ on rat granulosa cells. In addition to the growth-promoting actions of TGF-β plus FSH, TGF-β enhanced FSH-induced aromatase activity. Consequently, FSH plus TGF-β stimulates both the proliferation and aromatization capacity of rat granulosa cells. We propose that interactions between FSH, E₂ and TGF-β lead to the exponential increase in serum E₂ levels that occurs during the follicular phase of the cycle. Similarly, FSH stimulates the aromatization of exogenous androgens to estrogen by Sertoli cells isolated from immature rat testes, and there is a correlation between FSH-induced aromatization and mitotic activity. We have shown that FSH plus TGF-β stimulates DNA synthesis in Sertoli cells. Since E₂ increases the secretion of TGF-β by Sertoli cells, interactions between FSH, E₂ and TGF-β may provide the mitogenic stimulus for Sertoli cells during the prepubertal period. In summary, our findings suggest that the estrogen-induced growth of rat granulosa cells is mediated through the production of TGF-β, which acts as an autocrine regulator of proliferation. We also propose that the growth-promoting actions of FSH on Sertoli cells may depend upon a cascade series of events involving estrogens and TGF-β.     

The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space

We have examined the effect of transforming growth factor β₁ (TGF-β₁) and overexpression of the Smad4 gene on the phenotype of Car C, a ras mutated highly malignant spindle carcinoma cell line. TGF-β₁-treated Car C cells overexpressing Smad4 spread with a flattened morphology with membrane ruffles abundant in vinculin and show a reduction in their invasive abilities. TGF-β₁ treatment and overexpression of Smad4 also enhanced the production of PAI-1 measured by the activation of the p3TP-lux reporter gene containing a PAI-1-related promoter. This activation was abolished with a dominant-negative Smad4 construct. These results lead us to conclude that both TGF-β₁ and Smad4 overexpression reduce the invasive potential of Car C cells, probably via the Smad pathway.