Different Tyrosine Autophosphorylation Requirements in Fibroblast Growth Factor Receptor-1 Mediate Urokinase-Type Plasminogen Activator Induction and Mitogenesis

Among the seven tyrosine autophosphorylation sites identified in the intracellular domain of tyrosine kinase fibroblast growth factor receptor-1 (FGFR1), five of them are dispensable for FGFR1-mediated mitogenic signaling. The possibility of dissociating the mitogenic activity of basic FGF (FGF2) from its urokinase-type plasminogen activator (uPA)-inducing capacity both at pharmacological and structural levels prompted us to evaluate the role of these autophosphorylation sites in transducing FGF2-mediated uPA upregulation. To this purpose, L6 myoblasts transfected with either wild-type (wt) or various FGFR1 mutants were evaluated for the capacity to upregulate uPA production by FGF2. uPA was induced in cells transfected with wt-FGFR1, FGFR1-Y463F, -Y585F, -Y730F, -Y766F, or -Y583/585F mutants. In contrast, uPA upregulation was prevented in L6 cells transfected with FGFR1-Y463/583/585/730F mutant (FGFR1–4F) or with FGFR1-Y463/583/585/730/766F mutant (FGFR1–5F) that retained instead a full mitogenic response to FGF2; however, preservation of residue Y730 in FGFR1-Y463/583/585F mutant (FGFR1–3F) and FGFR1-Y463/583/585/766F mutant (FGFR1–4Fbis) allows the receptor to transduce uPA upregulation. Wild-type FGFR1, FGFR1–3F, and FGFR1–4F similarly bind to a 90-kDa tyrosine-phosphorylated protein and activate Shc, extracellular signal-regulated kinase (ERK)2, and JunD after stimulation with FGF2. These data, together with the capacity of the ERK kinase inhibitor PD 098059 to prevent ERK2 activation and uPA upregulation in wt-FGFR1 cells, suggest that signaling through the Ras/Raf-1/ERK kinase/ERK/JunD pathway is necessary but not sufficient for uPA induction in L6 transfectants. Accordingly, FGF2 was able to stimulate ERK1/2 phosphorylation and cell proliferation, but not uPA upregulation, in L6 cells transfected with the FGFR1-Y463/730F mutant, whereas the FGFR1-Y583/585/730F mutant was fully active. We conclude that different tyrosine autophosphorylation requirements in FGFR1 mediate cell proliferation and uPA upregulation induced by FGF2 in L6 cells. In particular, phosphorylation of either Y463 or Y730, dispensable for mitogenic signaling, represents an absolute requirement for FGF2-mediated uPA induction.

Epidermal Growth Factor Signaling and Mitogenesis in Plcg1 Null Mouse Embryonic Fibroblasts

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-γ1, a ubiquitous tyrosine kinase substrate. Plcg1−/− embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1+/+ embryos. The null cells do grow to a higher saturation density in serum-containing media, as their capacity to spread out is decreased compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1−/− cells respond equivalently to PLcg1+/+ cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-γ1 for many responses to growth factors.

Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3.

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3β protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3β protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF → Src → Stat3 → Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase.

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.

Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.

The effects of salbutamol, beclomethasone, and dexamethasone on fibronectin expression by cultured airway smooth muscle cells

Possible mechanisms of adverse drug effects in asthma include worsening of cellular hyperplasia and stimulation of extracellular matrix deposition. In this study, salbutamol, dexamethasone and beclomethasone were investigated to ascertain their ability to induce mitogenesis and stimulate fibronectin expression in cultured canine airway smooth muscle cells. In cells maintained in serum-free media for 72 h, salbutamol(1 nM-10 mu M) caused mitogenesis. The control cells had 2.57 +/- 0.34 x 10(5) cells per mi (mean +/- SEM, N = 13), while salbutamol (1 mu M) caused a maximal increase in cell number to 3.57 +/- 0.23 x 10(5) cells/ml (P < 0.01). In cells stimulated to replicate by addition of either fetal bovine serum or canine serum, no additional mitogenic effect of salbutamol was seen. Salbutamol did not have a detectable quantitative effect on fibronectin matrix expression. The glucocorticoids, beclomethasone and dexamethasone, significantly altered fibronectin expression by cultured airway smooth muscle cells. Beclomethasone increased fibronectin expression, while dexamethasone decreased expression.

Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases.

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1alpha, IL-1beta, CCL20/MIP-3alpha, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation.

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.

Insulin and insulin-like growth factor I receptors utilize different G protein signaling components.

We examined the role of heterotrimeric G protein signaling components in insulin and insulin-like growth factor I (IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the Galpha(i) inhibitor (pertussis toxin) or microinjection of the Gbetagamma inhibitor (glutathione S-transferase-betaARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Galpha(i) and Gbeta were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Galpha(i) increased concomitantly with a decrease in Gbeta association. No association of Galpha(i) was found with either the insulin or EGF receptor. Microinjection of anti-beta-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. beta-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Galpha(i), betagamma subunits, and beta-arrestin-1 all play a critical role in IGF-I mitogenic signaling. In contrast, neither metabolic, such as GLUT4 translocation, nor mitogenic signaling by insulin is dependent on these protein components. These results suggest that insulin receptors and IGF-IRs can function as G protein-coupled receptors and engage different G protein partners for downstream signaling.

Potential role for peroxisome proliferator activated receptor (PPAR) in preventing colon cancer.

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors involved in genetic control of many cellular processes. PPAR and PPAR have been implicated in colonic malignancy. Here we provide three lines of evidence suggesting an inhibitory role for PPAR in colorectal cancer development. METHODS: Levels of PPAR mRNA and protein in human colorectal cancers were compared with matched non-malignant mucosa using RNAse protection and western blotting. APC(Min)/+ mice were randomised to receive the PPAR activator methylclofenapate 25 mg/kg or vehicle for up to 16 weeks, and small and large intestinal polyps were quantified by image analysis. The effect of methylclofenapate on serum stimulated mitogenesis (thymidine incorporation), linear cell growth, and annexin V and propidium iodide staining were assessed in human colonic epithelial cells. RESULTS: PPAR (mRNA and protein) expression levels were significantly depressed in colorectal cancer compared with matched non-malignant tissue. Methylclofenapate reduced polyp area in the small intestine from 18.7 mm(2) (median (interquartile range 11.1, 26.8)) to 9.90 (4.88, 13.21) mm(2) (p=0.003) and in the colon from 9.15 (6.31, 10.5) mm(2) to 3.71 (2.71, 5.99) mm(2) (p=0.009). Methylclofenapate significantly reduced thymidine incorporation and linear cell growth with no effect on annexin V or propidium iodide staining. CONCLUSIONS: PPAR may inhibit colorectal tumour progression, possibly via inhibition of proliferation, and may be an important therapeutic target.

Metabolic effects of insulin and IGFs on gilthead sea bream (Sparus aurata) muscle cells.

Primary cultures of gilthead sea bream myocytes were performed in order to examine the relative metabolic function of insulin compared with IGF-I and IGF-II (insulin-like growth factors, IGFs) at different stages in the cell culture. In these cells, the in vitro effects of insulin and IGFs on 2-deoxyglucose (2-DG) and L-alanine uptake were studied in both myocytes (day 4) and small myotubes (day 9). 2-DG uptake in gilthead sea bream muscle cells was increased in the presence of insulin and IGFs in a time dependent manner and along with muscle cell differentiation. On the contrary, L-alanine uptake was also stimulated by insulin and IGFs but showed an inverse pattern, being the uptake higher in small myocytes than in large myotubes. The results of preincubation with inhibitors (PD-98059, wortmannin, and cytochalasin B) on 2-DG uptake indicated that insulin and IGFs stimulate glucose uptake through the same mechanisms, and evidenced that mitogenesis activator protein kinase (MAPK) and PI3K-Akt transduction pathways mediate the metabolic function of these peptides. In the same way, we observed that GLUT4 protein synthesis was stimulated in the presence of insulin and IGFs in gilthead sea bream muscle cells in a different manner at days 4 or 9 of the culture. In summary we describe here, for the first time, the effects of insulin and IGFs on 2-DG and L-alanine uptake in primary culture of gilthead sea bream muscle cells. We show that both MAPK and PI3K-Akt transduction pathways are needed in order to control insulin and IGFs actions in these cells. Moreover, changes in glucose uptake can be explained by the action of the GLUT4 transporter, which is stimulated in the presence of insulin and IGFs throughout the cell culture.

Biphasic modulation of insulin receptor substrate-1 during goitrogenesis

Insulin receptor substrate-1 (IRS-1) is the main intracellular substrate for both insulin and insulin-like growth factor I (IGF-I) receptors and is critical for cell mitogenesis. Thyrotropin is able to induce thyroid cell proliferation through the cyclic AMP intracellular cascade; however, the presence of either insulin or IGF-I is required for the mitogenic effect of thyroid-stimulating hormone (TSH) to occur. The aim of the present study was to determine whether thyroid IRS-1 content is modulated by TSH in vivo. Strikingly, hypothyroid goitrous rats, which have chronically high serum TSH levels (control, C = 2.31 ± 0.28; methimazole (MMI) 21d = 51.02 ± 6.02 ng/mL, N = 12 rats), when treated with 0.03% MMI in drinking water for 21 days, showed significantly reduced thyroid IRS-1 mRNA content. Since goiter was already established in these animals by MMI for 21 days, we also evaluated IRS-1 expression during goitrogenesis. Animals treated with MMI for different periods of time showed a progressive increase in thyroid weight (C = 22.18 ± 1.21; MMI 5d = 32.83 ± 1.48; MMI 7d = 31.1 ± 3.25; MMI 10d = 33.8 ± 1.25; MMI 14d = 45.5 ± 2.56; MMI 18d = 53.0 ± 3.01; MMI 21d = 61.9 ± 3.92 mg, N = 9-15 animals per group) and serum TSH levels (C = 1.57 ± 0.2; MMI 5d = 9.95 ± 0.74; MMI 7d = 10.38 ± 0.84; MMI 10d = 17.72 ± 1.47; MMI 14d = 25.65 ± 1.23; MMI 18d = 35.38 ± 3.69; MMI 21d = 31.3 ± 2.7 ng/mL, N = 9-15 animals per group). Thyroid IRS-1 mRNA expression increased progressively during goitrogenesis, being significantly higher by the 14th day of MMI treatment, and then started to decline, reaching the lowest values by the 21st day, when a significant reduction was detected. In the liver of these animals, however, a significant decrease of IRS-1 mRNA was detected after 14 days of MMI treatment, a mechanism probably involved in the insulin resistance that occurs in hypothyroidism. The increase in IRS-1 expression during goitrogenesis may represent an important event associated with the increased rate of cell mitosis promoted by TSH and indicates that insulin and IGF-I are important co-mitogenic factors in vivo, possibly acting through the activation of IRS-1.

Biological effects of resveratrol on skeletal muscle cells

Resveratrol, a polyphenol found in red wine, has been reported to have antithrombotic, antiatherogenic, and anticancer properties both in vitro and III VIVO. However, possible antidiabetic properties of resveratrol have not been examined. The objective of this study was to investigate the direct effects of resveratrol on basal and insulin-stimulated glucose uptake and to elucidate its mechanism of action in skeletal muscle cells. In addition, the effects of resveratrol on basal and insulin- stimulated amino acid transport and mitogenesis were also examined. Fully differentiated L6 rat skeletal muscle cells were incubated with resveratrol concentrations ranging from 1 to 250 IlM for 15 to 120 min. Maximum stimulation, 201 ± 8.90% of untreated control, (p<0.001), of2eH] deoxy- D- glucose (2DG) uptake was seen with 100 IlM resveratrol after 120 min. Acute, 30 min, exposure of the cells to 100 nM insulin stimulated 2DG uptake to 226 ± 12.52% of untreated control (p<0.001). This appears to be a specific property of resveratrol that is not shared by structurally similar antioxidants such as quercetin and rutin, both of which did not have any stimulatory effect. Resveratrol increased the response of the cells to submaximal insulin concentrations but did not alter the maximum insulin response. Resveratrol action did not require insulin and was not blocked by the protein synthesis inhibitor cycloheximide. L Y294002 and wortmannin, inhibitors of PI3K, abolished both insulin and resveratrolstimulated glucose uptake while phosphorylation of AktlPKB, ERK1I2, JNK1I2, and p38 MAPK were not increased by resveratrol. Resveratrol did not stimulate GLUT4 transporter translocation in GLUT4cmyc overexpressing cells, in contrast to the significant translocation observed with insulin. Furthermore, resveratrol- stimulated glucose transport was not blocked by the presence of the protein kinase C (PKC) inhibitors BIMI and G06983. Despite that, resveratrol- induced glucose transport required an intact actin network, similar to insulin. In contrast to the stimulatory effect seen with resveratrol for glucose transport, e4C]methylaminoisobutyric acid (MeAIB) transport was inhibited. Significant reduction of MeAIB uptake was seen only with 100uM resveratrol (74.2 ± 6.55% of untreated control, p<0.05), which appeared to be maximum. In parallel experiments, insulin (100 nM, 30 min) increased MeAIB transport by 147 ± 5.77% (p<0.00l) compared to untreated control. In addition, resveratrol (100 JlM, 120 min) completely abolished insulin- stimulated amino acid transport (103 ± 7.35% of untreated control,p>0.05). Resveratrol also inhibited cell proliferation in L6 myoblasts with maximal inhibition of eH]thymidine incorporation observed with resveratrol at 50 J.LM after 24 hours (8 ± 1.59% of untreated control, pmitogenesis.