Mitogenic effect of transforming growth factor beta 1 on human fibroblasts involves the induction of platelet-derived growth factor alpha receptors

Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), potent modulators of mesenchymal cell growth and differentiation, are often colocalizable in vivo. Previous in vitro studies in fibroblastic cell lines have shown variable, even antagonistic effects of TGF-beta on the mitogenic action of PDGF. This study demonstrates that in diploid human dermal fibroblasts, TGF-beta 1 is weakly mitogenic in the absence of serum or purified growth factors, and that TGF-beta 1 potentiates DNA synthesis in PDGF-stimulated fibroblasts with delayed kinetics when compared to stimulation with PDGF alone. TGF-beta 1 enhances mitogenic potency of all three PDGF isoforms and increases receptor binding of both 125I PDGF-AA and 125I PDGF-BB, consistent with the increased expression of the alpha type PDGF receptor. The induction of PDGF alpha receptor subunits by TGF-beta may play a role in enhancing the proliferative potential of human fibroblasts in certain physiologic and pathologic conditions.     

NAD(P)H oxidase-derived reactive oxygen species regulate angiotensin-II induced adventitial fibroblast phenotypic differentiation

Phenotypic differentiation of adventitial fibroblasts into myofibroblasts is an essential feature of vascular remodeling. The present study was undertaken to test the hypothesis that reactive oxygen species (ROS) are involved in rat adventitial fibroblast differentiation to myofibroblast. Activation of alpha-smooth muscle actin (alpha-SMA) was used as a marker of myofibroblast. Angiotensin II increased intracellular ROS in adventitial fibroblasts that was completely inhibited by the free radical scavenger NAC, the NAD(P)H oxidase inhibitor DPI, and transfection of antisense gp91phox oligonucleotides. Myofibroblast differentiation was prevented by inhibition of ROS generation with DPI, NAC, and antisense gp91phox as shown by decreased expression of alpha-SMA. Angiotensin II rapidly induced phosphorylation of p38 MAPK and JNK, both of which were inhibited by DPI, NAC, antisense gp91phox, and the selective AT1 receptor antagonist, losartan. Inhibiting p38MAPK with SB202190 or JNK with SP600125 also reduced angiotensin II-induced alpha-SMA expression. These findings demonstrate that angiotensin II induces adventitial fibroblast differentiation to myofibroblast via a pathway that involves NADPH oxidase generation of ROS and activation of p38MAPK and JNK pathways.     

Transforming growth factor Beta1 induction of tissue inhibitor of metalloproteinases 3 in articular chondrocytes is mediated by reactive oxygen species

Transforming growth factor beta1 (TGF-beta1) stimulates cartilage extracellular matrix synthesis but, in excess, evokes synovial inflammation, hyperplasia, and osteophyte formation in arthritic joints. TGF-beta1 induces tissue inhibitor of metalloproteinases 3 (TIMP-3), an inhibitor of cartilage-damaging matrix metalloproteianases and aggrecanases. We investigated the role of reactive oxygen species (ROS) in TIMP-3 induction by TGF-beta1. In primary human and bovine chondrocytes, ROS scavenger and antioxidant N-acetylcysteine (NAC) inhibited TGF-beta1-induced TIMP-3 mRNA and protein increases. Ebselen and ascorbate also reduced this induction. TGF-beta1 time-dependently induced ROS production that was suppressed by NAC. Hydrogen peroxide, a ROS, induced TIMP-3 RNA. The TIMP-3 increase induced by TGF-beta1 was partly Smad2-dependent. TGF-beta1-stimulated Smad2 phosphorylation was inhibited by NAC. Reduced glutathione and L-cysteine also blocked Smad2 and TIMP-3 induction by TGF-beta1, whereas a nonthiol, N-acetylalanine, did not. Smad2 was not activated by H2O2. Smad2 phosphorylation was independent, and TIMP-3 expression was dependent, on new protein synthesis. TGF-beta-stimulated ERK and JNK phosphorylation was also inhibited by NAC. However, inhibitory actions of NAC were not mediated by ERK activation. Thus, ROS mediate TGF-beta1-induced TIMP-3 gene expression. Blocking TGF-beta1-induced gene expression by modulating cellular redox status with thiols can be potentially beneficial for treating arthritic and other disorders caused by excessive TGF-beta1.     

Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells

Transforming growth factor-beta1 (TGF-beta1) is abundantly expressed in pulmonary hypertension, but its effect on the pulmonary circulation remains unsettled. We studied the consequences of TGF-beta1 stimulation on freshly isolated human pulmonary artery smooth muscle cells (HPASMC). TGF-beta1 initially promoted differentiation, with upregulated expression of smooth muscle contractile proteins. TGF-beta1 also induced expression of Nox4, the only NAD(P)H oxidase membrane homolog found in HPASMC, through a signaling pathway involving Smad 2/3 but not mitogen-activated protein (MAP) kinases. TGF-beta1 likewise increased production of reactive oxygen species (ROS), an effect significantly reduced by the NAD(P)H oxidase flavoprotein inhibitor diphenylene iodonium (DPI) and by Nox4 siRNAs. In the absence of TGF-beta1, Nox4 was present in freshly cultured cells but progressively lost with each passage in culture, paralleling a decrease in ROS production by HPASMC over time. At a later time point (72 h), TGF-beta1 promoted HPASMC proliferation in a manner partially inhibited by Nox4 small interfering RNA and dominant negative Smad 2/3, indicating that TGF-beta1 stimulates HPASMC growth in part by a redox-dependent mechanism mediated through induction of Nox4. HPASMC activation of the MAP kinases ERK1/2 was reduced by the NAD(P)H oxidase inhibitors DPI and 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that TGF-beta1 may facilitate proliferation by upregulating Nox4 and ROS production, with transient oxidative inactivation of phosphatases and augmentation of growth signaling cascades. These findings suggest that Nox4 is the relevant Nox homolog in HPASMC. This is the first observation that TGF-beta1 regulates Nox4, with important implications for mechanisms of pulmonary vascular remodeling.     

Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1.

Mitogenic growth factors and transforming growth factor beta1 (TGF-beta1) induce the generation of reactive oxygen species (ROS) in nonphagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF-beta1 to activate a cell surface-associated NADH:flavin:O(2) oxidoreductase (NADH oxidase) that generates extracellular H(2)O(2). In this study, we compared the ROS-generating enzymatic systems activated by mitogenic growth factors and TGF-beta1 with respect to the primary reactive species produced (O(2)(.-) vs. H(2)O(2)), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF-BB, FGF-2, and TGF-alpha (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O(2)(.-) without detectable NADH oxidase activity or extracellular H(2)O(2) release. In contrast, TGF-beta1 does not stimulate intracellular O(2)(.-) production and the delayed induction of extracellular H(2)O(2) release is not associated with O(2)(.-) production. Expression of dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O(2)(.-) generation but has no effect on TGF-beta1-induced NADH oxidase activation/H(2)O(2) production. These results demonstrate that there are at least two distinctly different ROS-generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF-beta1 via Ras-dependent and -independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/cytokines may have different biological effects depending on the primary reactive species generated and site of production.     

bFGF Promotes the Migration of Human Dermal Fibroblasts under Diabetic Conditions through Reactive Oxygen Species Production via the PI3K/Akt-Rac1- JNK Pathways

Fibroblasts play a pivotal role in the process of cutaneous wound repair, whereas their migratory ability under diabetic conditions is markedly reduced. In this study, we investigated the effect of basic fibroblast growth factor (bFGF) on human dermal fibroblast migration in a high-glucose environment. bFGF significantly increased dermal fibroblast migration by increasing the percentage of fibroblasts with a high polarity index and reorganizing F-actin. A significant increase in intracellular reactive oxygen species (ROS) was observed in dermal fibroblasts under diabetic conditions following bFGF treatment. The blockage of bFGF-induced ROS production by either the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) almost completely neutralized the increased migration rate of dermal fibroblasts promoted by bFGF. Akt, Rac1 and JNK were rapidly activated by bFGF in dermal fibroblasts, and bFGF-induced ROS production and promoted dermal fibroblast migration were significantly attenuated when suppressed respectively. In addition, bFGF-induced increase in ROS production was indispensable for the activation of focal adhesion kinase (FAK) and paxillin. Therefore, our data suggested that bFGF promotes the migration of human dermal fibroblasts under diabetic conditions through increased ROS production via the PI3K/Akt-Rac1-JNK pathways.     

TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.     

Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture

A transforming growth factor-beta (TGF-beta) found in platelets strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin plus EGF or by hepatocyte growth factor (HGF) from rat platelets, but not the syntheses of secretory and intracellular proteins by the cells. TGF-beta had no cytotoxic effect, as judged by phase-contrast microscopic examination of the cell morphology. The inhibition of DNA synthesis by TGF-beta was correlated with marked decrease in the labeling index. TGF-beta did not inhibit growth of hepatoma cell line. These findings indicate that TGF-beta is a strong growth inhibitor of adult rat hepatocytes and may block their shift from the G1 phase to the S phase. The physiological role of TGF-beta in inhibiting growth of adult hepatocytes during liver regeneration is discussed.     

Transforming growth factor-beta 1 inhibitory effect of platelet-derived growth factor-induced signal transduction on human bone marrow fibroblasts: possible involvement of protein phosphatases.

Transforming growth factor-beta 1 (TGF-beta 1) is a potent growth inhibitor for many cell types. On fibroblasts, TGF-beta 1 has been shown to inhibit human platelet-derived growth factor (PDGF)-induced mitogenicity. The mechanism implicated in this growth inhibition is unknown. In this work, we show on human bone marrow fibroblasts that TGF-beta 1, which inhibited PDGF-BB mitogenicity, was able to block PDGF-BB-induced early events such as polyphosphoinositide (PtdIns 4,5-P2, PtdIns 4-P, and PtdIns) breakdown and Ins 1,4,5-P3 formation. No significant modification by TGF-beta 1 of PDGF-BB binding (n1 = 200,000 vs. n2 = 195,000 sites per cell with TGF-beta 1; Kd1 = Kd2 = 0.5 x 10(-9) M) and of internalization kinetics was observed. In addition, TGF-beta 1 was shown to inhibit PDGF-BB receptor autophosphorylation either in intact cells or in partially isolated membranes and to partially inhibit PDGF-R tyrosine kinase activity. Since a dephosphorylation mechanism through protein phosphatases could be implicated, we used okadaic acid, a potent inhibitor of type 1 and 2A serine/threonine phosphatases and showed that okadaic acid restored PDGF-receptor autophosphorylation on tyrosine residues. Based on these data, we suggest that an alternative regulatory mechanism of PDGF tyrosine phosphorylation seems to involve serine/threonine phosphatase activation.     

Activation of NADPH oxidase by transforming growth factor-beta in hepatocytes mediates up-regulation of epidermal growth factor receptor ligands through a nuclear factor-kappaB-dependent mechanism

The TGF-beta (transforming growth factor-beta) induces survival signals in foetal rat hepatocytes through transactivation of EGFR (epidermal growth factor receptor). The molecular mechanism is not completely understood, but both activation of the TACE (tumour necrosis factor alpha-converting enzyme)/ADAM17 (a disintegrin and metalloproteinase 17; one of the metalloproteases involved in shedding of the EGFR ligands) and up-regulation of TGF-alpha and HB-EGF (heparin-binding epidermal growth factor-like growth factor) appear to be involved. In the present study, we have analysed the molecular mechanisms that mediate up-regulation of the EGFR ligands by TGF-beta in foetal rat hepatocytes. The potential involvement of ROS (reactive oxygen species), an early signal induced by TGF-beta, and the existence of an amplification loop triggered by initial activation of the EGFR, have been studied. Results indicate that DPI (diphenyleneiodonium) and apocynin, two NOX (NADPH oxidase) inhibitors, and SB431542, an inhibitor of the TbetaR-I (TGF-beta receptor I), block up-regulation of EGFR ligands and Akt activation. Different members of the NOX family of genes are expressed in hepatocytes, included nox1, nox2 and nox4. TGF-beta up-regulates nox4 and increases the levels of Rac1 protein, a known regulator of both Nox1 and Nox2, in a TbetaR-I-dependent manner. TGF-beta mediates activation of the nuclear factor-kappaB pathway, which is inhibited by DPI and is required for up-regulation of TGF-alpha and HB-EGF. In contrast, EGFR activation is not required for TGF-beta-induced up-regulation of those ligands. Considering previous work that has established the role of ROS in apoptosis induced by TGF-beta in hepatocytes, the results of the present study indicate that ROS might mediate both pro- and anti-apoptotic signals in TGF-beta-treated cells.     

Growth factor modulation of mitogenic responses and proteoglycan synthesis by human periodontal fibroblasts

In order to understand the relationship between specific growth factors and matrix synthesis by periodontal cells, we have investigated the effects of platelet-derived growth factor BB (PDGF-BB), insulin-like growth factor-I (IGF-1), and growth hormone on DNA and proteoglycan synthesis by cultured human gingival and periodontal ligament fibroblasts in vitro. PDGF-BB and IGF-1, but not growth hormone, were mitogenic for both periodontal ligament fibroblasts and gingival fibroblasts, although the periodontal ligament cells responded more strongly. The mitogenic response was accompanied by alterations in expression of matrix proteoglycan mRNA. For both the gingival and periodontal ligament cells, there was a decrease in mRNA for decorin and an increase in mRNA for versican following exposure to IGF-1 and PDGF-BB. Although no change was seen in response to PDGF, biglycan mRNA level was increased by IGF-1 in periodontal ligament fibroblasts. With the gingival fibroblats, biglycan mRNA levels were unaffected by IGF-1, PDGF-BB, or growth hormone. These findings suggest variable responses of fibroblasts to growth factors depending upon anatomical site within the periodontium. Moreover, there appears to be a correlation between cell proliferation and the types of proteoglycan synthesised with decorin expression being suppressed, and versican being increased during fibroblast proliferation. J. Cell. Physiol. 174:353–361, 1998. © 1998 Wiley-Liss, Inc.     

Reactive oxygen species mediate Endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 signaling, as well as protein synthesis, in vascular smooth muscle cells

Reactive oxygen species (ROS) have been shown to mediate the effects of several growth factors and vasoactive peptides, such as epidermal growth factor, platelet-derived growth factor, and angiotensin II (AII). Endothelin-1 (ET-1) is a vasoactive peptide which also exhibits mitogenic activity in vascular smooth muscle cells (VSMCs), and is believed to contribute to the pathogenesis of vascular abnormalities such as atherosclerosis, hypertension, and restenosis after angioplasty. However, a possible role for ROS generation in mediating the ET-1 response on extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB), and protein tyrosine kinase 2 (Pyk2), key components of the growth-promoting and proliferative signaling pathways, has not been examined in detail. Our aim was to investigate the involvement of ROS in ET-1-mediated activation of ERK1/2, PKB, and Pyk2 in A-10 VSMCs. ET-1 stimulated ERK1/2, PKB, and Pyk2 phosphorylation in a dose- and time-dependent manner. Pretreatment of A-10 VSMCs with diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate oxidase, attenuated ET-1-enhanced ERK1/2, PKB, and Pyk2 phosphorylation. In addition, in parallel with an inhibitory effect on the above signaling components, DPI also blocked ET-1-induced protein synthesis. ET-1 was also found to increase ROS production, which was suppressed by DPI treatment. N-Acetylcysteine, a ROS scavenger, exhibited a response similar to that of DPI and inhibited ET-1-stimulated ERK1/2, PKB, and Pyk2 phosphorylation. These results demonstrate that ROS are critical mediators of ET-1-induced signaling events linked to growth-promoting proliferative and hypertrophic pathways in VSMCs.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

Platelet-derived growth factor and transforming growth factor-beta enhance tissue repair activities by unique mechanisms

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these growth factors might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and fibroblasts as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (20 micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to 212% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and fibroblasts to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in fibroblasts in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of fibroblasts and granulation tissue through day 21, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and fibroblasts from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney fibroblasts. The results suggest that TGF-beta transiently attracts fibroblasts into the wound and may stimulate collagen synthesis directly. In contrast, PDGF is a more potent chemoattractant for wound macrophages and fibroblasts and may stimulate these cells to express endogenous growth factors, including TGF-beta, which, in turn, directly stimulate new collagen synthesis and sustained enhancement of wound healing over a more prolonged period of time.     

Ethanol potentiates the mitogenic effects of sphingosine 1-phosphate by a zinc- and calcium-dependent mechanism in fibroblasts.

In mouse embryo NIH 3T3 fibroblasts, ethanol (60-80 mM) was found to enhance the stimulatory effects of sphingosine 1-phosphate (S1P) on both DNA synthesis and cell proliferation. Well-detectable potentiating effects of ethanol on S1P-induced mitogenesis required the presence of calcium (>1 mM) and zinc (20-40 microM) in the incubation medium. The amphibian tetrapeptide bombesin, which is known to mobilize intracellular calcium in fibroblasts, had no effect alone, but it approximately doubled the combined stimulatory effects of ethanol and S1P on DNA synthesis. The synergistic mitogenic effects of ethanol and S1P were also slightly enhanced, rather than inhibited, by the alcohol dehydrogenase inhibitor 4-methylpyrazole (5 mM). Of the various growth regulatory enzymes examined, ethanol detectably enhanced the stimulatory effects of S1P on the phosphosphorylation (activation) of p42/p44 mitogen-activated protein (MAP) kinases, but not of p38 MAP kinase. Cotreatment of fibroblasts with ethanol for 10 min also enhanced the stimulatory effects of S1P on the activities of c-Raf-1 kinase and p70 S6 kinase, but neither S1P nor ethanol had effects on phosphatidylinositol 3'-kinase and Akt/PKB kinase activities. Ethanol-plus-S1P-induced DNA synthesis was partially inhibited by both PD 98059 (50 microM) and rapamycin (10 nM), inhibitors of p42/p44 MAP kinase kinase and mTOR/p70 S6 kinases, respectively. The results indicate that in NIH 3T3 fibroblasts, ethanol can enhance the mitogenic effects of S1P by a zinc- and calcium-dependent mechanism involving both the rapamycin-sensitive p70 S6 kinase-dependent and the c-Raf-1/MAP kinase-dependent growth regulatory pathways.     

Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-beta

Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway, but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-beta is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-beta1 mRNA and protein to a greater extent than nonasthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-beta1 stimulated the growth of nonconfluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-beta1 on ASM cells was inhibited by selective inhibitors of TGF-beta receptor I kinase (SD-208), phosphatidylinositol 3-kinase (PI3K, LY-294002), ERK (PD-98059), JNK (SP-600125), and NF-kappaB (AS-602868). On the other hand, p38 MAPK inhibitor (SB-203580) augmented TGF-beta1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector-expressing Smad4, Smad7, or dominant-negative Smad3 and found no involvement of these Smads in TGF-beta1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-beta1-induced proliferation. Our findings suggest that TGF-beta1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK, and JNK, whereas p38 MAPK is a negative regulator. NF-kappaB is also involved in the TGF-beta1 mitogenic signaling, but Smad pathway does not appear important.     

Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts.

The effect of various growth factors on the synthesis of hyaluronan in human fibroblasts was investigated. When tested in medium containing 0.5% fetal calf serum, platelet-derived growth factor (PDGF)-BB was found to stimulate hyaluronan synthesis; the maximal response was equal to or higher than that obtained with 10% fetal calf serum. PDGF-AA gave only a limited effect, indicating that the stimulatory effect of PDGF on hyaluronan synthesis was mainly transduced via the B-type PDGF receptor. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 1 also stimulated hyaluronan synthesis; their effects were less than that of PDGF-BB, but combinations of factors produced potent stimulatory effects on hyaluronan synthesis. All factors stimulated hyaluronan synthesis in sparse as well as dense cultures. The effects of the factors on hyaluronan synthesis did not correlate with their mitogenic activities; PDGF-BB, EGF and bFGF are equipotent mitogens, but PDGF-BB had a much more potent effect on hyaluronan synthesis, and TGF-beta actually inhibits the growth of fibroblasts under the conditions of the assay.     

Involvement of reactive oxygen species in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm,Bombyx mori

In the present study, the possible involvement of reactive oxygen species (ROS) in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis of Bombyx mori prothoracic glands (PGs) was investigated. Results showed that PTTH treatment resulted in a rapidly transient increase in the intracellular ROS concentration, as measured using 2′,7′-dichlorofluorescin diacetate (DCFDA), an oxidation-sensitive fluorescent probe. The antioxidant, N-acetylcysteine (NAC), abolished PTTH-induced increase in fluorescence. Furthermore, PTTH-induced ROS production was partially inhibited by the NAD(P)H oxidase inhibitor, apocynin, indicating that NAD(P)H oxidase is one of the sources for PTTH-stimulated ROS production. Four mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenylene iodonium (DPI)) significantly attenuated ROS production induced by PTTH. These data suggest that the activity of complexes I and III in the electron transport chain and the mitochondrial inner membrane potential (ΔΨ) contribute to PTTH-stimulated ROS production. In addition, PTTH-stimulated ecdysteroidogenesis was greatly inhibited by treatment with either NAC or mitochondrial inhibitors (rotenone, antimycin A, FCCP, and DPI), but not with apocynin. These results indicate that mitochondria-derived, but not membrane NAD(P)H oxidase-mediated ROS signaling, is involved in PTTH-stimulated ecdysteroidogenesis of PGs in B. mori.     

Platelet-derived growth factor modulates rat vascular smooth muscle cell responses on laminin-5 via mitogen-activated protein kinase-sensitive pathways

BACKGROUND: A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM. RESULTS: Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-beta1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-beta1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-beta1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did. CONCLUSION: These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis.