Platelet-derived growth factor (PDGF) initiates replication of density-arrested BALB/c-3T3 mouse cells by rendering them "competent" to respond to factors contained in plasma. Treatment of quiescent cells with PDGF rapidly stimulates the preferential synthesis of several cytoplasmic proteins (molecular weights 29,000 to 70,000). Four of these proteins were noted within 1.5 hr of PDGF addition and one (pI) within 40 min. Inhibitors of RNA synthesis prevented the synthesis of these proteins. Both the synthesis of pI and the stimulation of DNA synthesis displayed a similar dose response to PDGF concentration. Pituitary fibroblast growth factor, which also induces competence, stimulated pI and pII synthesis. Plasma, epidermal growth factor, or insulin, which do not induce competence, did not stimulate selective synthesis of these proteins. A transformed variant of BALB/c-3T3 cells, which has retained the growth requirement for plasma factors but lost the requirement for PDGF, synthesizes these PDGF-modulated proteins constitutively.

Three different receptor tyrosine kinases, epidermal growth factor (EGF), c-erbB-2/neu, and platelet-derived growth factor (PDGF) receptors, have been found to be present in the mouse mammary epithelial cell line HC11. We have investigated the consequences of receptor activation on the growth and differentiation of HC11 cells. HC11 cells are normal epithelial cells which maintain differentiation-specific functions. Treatment of the cells with the lactogenic hormones glucocorticoids and prolactin leads to the expression of the milk protein beta-casein. Activation of EGF receptor has a positive effect on cell growth and causes the cells to become competent for the lactogenic hormone response. HC11 cells respond optimally to the lactogenic hormone mixture and synthesize high levels of beta-casein only if they have been kept previously in a medium containing EGF. Transfection of HC11 cells with the activated rat neuT receptor results in the acquisition of competence to respond to the lactogenic hormones even if the cells are grown in the absence of EGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC11 cells. Cells kept only in PDGF do not become competent for lactogenic hormone induction. The results show that activation of the structurally related EGF and c-erbB-2/neu receptors, but not the PDGF receptor, allows the HC11 cells to subsequently respond optimally to lactogenic hormones.

Although leptin induces fibrotic activity in hepatic stellate cells (HSCs), the mechanisms are not entirely understood. To investigate the potential role of reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH) and reactive oxygen species (ROS) in leptin signaling in HSCs, we analyzed leptin-induced intracellular signaling pathways in primary wild-type (WT), p47(phox(-/-) ), and signal transducer and activator of transcription protein 3 (STAT3)-deleted HSCs. Leptin-stimulated ROS production was attenuated in human and mouse HSCs by the NADPH oxidase inhibitor diphenylene-iodonium (DPI) and in HSCs lacking the NADPH component p47(phox). Leptin-induced phosphorylation of extracellular signal-regulated kinase (ERK) and AKT, but not of STAT3, was blocked by NADPH oxidase inhibition. Moreover, leptin-induced ROS production was inhibited by the Janus kinase (JAK) inhibitor, AG490, but normal ROS production was observed in STAT3-deleted HSCs. Pharmacologic or genetic inhibition of NADPH in HSCs not only resulted in a reduction of leptin-mediated HSC proliferation but also reduced the leptin-mediated up-regulation of the fibrogenic markers collagen alpha1(I) and alpha-smooth muscle actin and of the inflammatory mediators monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), and macrophage inflammatory protein 2 (MIP-2). In vivo, leptin enhanced chemokine expression induced by chemokine (C-C motif) ligand 4 (CCl(4)) in WT mice, but a blunted response was observed in p47(phox-/-) mice. In conclusion, NADPH oxidase is a crucial mediator of proliferative, fibrogenic, and inflammatory actions of leptin. Leptin-induced NADPH oxidase acts downstream of JAK activation but is independent of STAT3. Our results, in conjunction with previous studies on angiotensin II and platelet-derived growth factor (PDGF), place NADPH in the center of the fibrogenic signaling response in HSCs and demonstrate its potential role as a pharmacological target for antifibrotic therapies.

OBJECTIVE

Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular growth and recruitment of perivascular mural cells. The purpose of the present study is to investigate the signalling events underlying the stimulation of vasculogenesis of mouse embryonic stem (ES) cells by PDGF-BB.

RESULTS

PDGF-BB increased vascular sprouting and branching of capillary-like structures in embryoid bodies as evaluated by computer-assisted analysis of CD31-positive cell structures. It also activated extracellular-regulated kinase 1,2 (ERK1,2) and c-Jun N-terminal kinase but not p38 mitogen-activated protein kinase or PI 3-kinase. Microfluorometric analysis of fluo-4 fluorescence revealed that treatment with PDGF-BB raised intracellular Ca(2+) levels in differentiating ES cells expressing the PDGF receptor beta, an effect that was abolished in the presence of the intracellular Ca(2+) chelator BAPTA. Furthermore, PDGF-BB raised reactive oxygen species (ROS) levels in embryoid bodies as evaluated using the redox-sensitive dye H(2)DCF-DA. ROS generation was blunted in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin as well as in the presence of BAPTA, suggesting that ROS generation is regulated by intracellular Ca(2+) transients. The stimulation of vasculogenesis of ES cells upon treatment with PDGF-BB was significantly inhibited by the ERK1,2 inhibitor U0126, the NADPH oxidase inhibitors DPI, apocynin, 4-(2-aminoethyl)benzenesulfonylfluoride and VAS2870, the free radical scavengers vitamin E, and N-(2-mercaptopropionyl)glycin as well as by BAPTA.

CONCLUSIONS

Our data demonstrate that the pro-vasculogenic effects of PDGF-BB are mediated by Ca(2+)-induced ROS generation, resulting in the activation of an ERK1,2-mediated signal transduction cascade.

4-hydroxynonenal is a major product of lipid peroxidation. It was firstly studied under the point of view of its toxicity, as it is an easily diffusable substance, thought to be able to explain the "far damages" seen in conditions of increased lipid peroxidation. Really, when used at concentration from 10 microM to 1 mM, usually referred to as high concentrations, the aldehyde is able to produce strong inhibitions of several enzymatic activities. When used, however, at concentration of 1 microM or lower, it displays a lot of activities regarding especially cell multiplication and differentiation. As the concentrations indicated above are usually found in normal tissues, these effects may be considered as physiological. As a low level of lipid peroxidation exists in normal tissues, the aldehyde displays signalling activities in normal cells. Among them, it is to consider the stimulation of neutrophil chemotaxis, the strong activation of plasmamembrane adenylate kinase, the strong activation of membrane phospholipase C, both in hepatocytes and neutrophils, the block in the expression of the oncogene c-myc in human leukemic cells, accompanied by differentiation of the same cells, the effects on the cyclins and the activity of E2F transcription factor, the strong increase of the expression of the gene for procollagen alfa1(I), occurring due to the activation of the c-jun/junkinases/AP-1 pathway. Moreover, it is able to block the activity of the PDGF-beta receptor. The last facts allow to think that a hydroxynonenal pathway works in the production of fibrosis.

The ability of dopamine D(4) and D(2) receptors to activate extracellular signal-regulated kinases (ERKs) 1 and 2 was compared using Chinese hamster ovary (CHO-K1) cells transfected with D(4.2), D(4.4), D(4.7), and D(2L) receptors. Dopamine stimulation of D(4) or D(2L) receptors produced a transient, dose-dependent increase in ERK1/2 activity. Receptor-specific activation of the ERK mitogen-activated protein kinase (MAPK) pathway was confirmed using the D(2)-like receptor-selective agonist quinpirole, whereas the specific antagonist haloperidol blocked activation. MAPK stimulation was dependent on a pertussis-toxin-sensitive G protein (G(i/o)). trans-Activation of the platelet-derived growth factor (PDGF) receptor was an essential step in D(4) and D(2L) receptor-induced MAPK activation. PDGF receptor-selective tyrosine kinase inhibitors tyrphostin A9 and AG1295 abolished or significantly inhibited ERK1/2 activation by D(4) and D(2L) receptors. Dopamine stimulation of the D(4) receptor also produced a rapid increase in tyrosine phosphorylation of the PDGF receptor-beta. The Src-family tyrosine kinase inhibitor PP2 blocked MAPK activation by dopamine; however, this drug was also found to inhibit PDGF-BB-stimulated ERK activity and autophosphorylation of the PDGF receptor-beta. Downstream signaling pathways support the involvement of a receptor tyrosine kinase. The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, protein kinase C inhibitors GF109203X and Calphostin C, dominant-negative RasN17, and the MEK inhibitor PD98059 significantly attenuated or abolished activation of MAPK by dopamine D(4) and D(2L) receptors. Our results indicate that D(4) and D(2L) receptors activate the ERK kinase cascade by first mobilizing signaling by the PDGF receptor, followed by the subsequent activation of ERK1/2 by pathways associated with this receptor tyrosine kinase.

Valve interstitial cells (VICs) are responsible for maintaining the structural integrity and dynamic behaviour of the valve. Telocytes (TCs), a peculiar type of interstitial cells, have been recently identified by Popescu's group in epicardium, myocardium and endocardium (visit www.telocytes.com). The presence of TCs has been identified in atria, ventricles and many other tissues and organ, but not yet in heart valves. We used transmission electron microscopy and immunofluorescence methods (double labelling for CD34 and c-kit, or vimentin, or PDGF Receptor-β) to provide evidence for the existence of TCs in human heart valves, including mitral valve, tricuspid valve and aortic valve. TCs are found in both apex and base of heart valves, with a similar density of 27-28 cells/mm(2) in mitral valve, tricuspid valve and aortic valve. Since TCs are known for the participation in regeneration or repair biological processes, it remains to be determined how TCs contributes to the valve attempts to re-establish normal structure and function following injury, especially a complex junction was found between TCs and a putative stem (progenitor) cell.

Vimentin exhibits a complex pattern of developmental and tissue-specific expression regulated by such growth factors as TGFbeta1, PDGF, FGF, EGF and cytokines. Vimentin is expressed in the more migratory, mesenchymal cell and its expression is often down-regulated to make way for tissue-specific intermediate filaments proteins such as desmin in muscle. Here, we suggest a mechanism to explain how TGFbeta1 contributes to the up-regulation of vimentin expression while blocking myogenesis. TGFbeta1 binds to serine/threonine kinase receptors resulting in the phosphorylation of Smad2 and Smad3, followed by formation of a heteromeric complex with Smad4. The translocation of this complex to the nucleus modulates transcription of selected genes such as vimentin. However, the vimentin gene lacks a consensus TGFbeta1 response element. By transient transfection analysis of vimentin's various promoter elements fused to the CAT reporter gene, we have determined that tandem AP-1 sites surrounded by GC-boxes are required for TGFbeta1 induction. Mutations within this region eliminated the ability of Smad3 to induce reporter gene expression. DNA precipitation and ChIP assays suggest that c-Jun, c-Fos, Smad3 and Sp1/Sp3 interact over this region, but this interaction changes during myogenesis with TGFbeta1 induction.

BACKGROUND

Alternative treatment options are needed for advanced neuroblastoma patients because their prognosis remains poor after intensive chemotherapy. Neuroblastoma cells express platelet-derived growth factor (PDGF), stem cell factor (SCF), and vascular endothelial growth factor (VEGF) and their respective receptors, PDGFR, c-Kit, and Flk-1. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the tyrosine kinase activities of c-Kit and PDGFR, on the growth of neuroblastoma cells in vivo and in vitro.

METHODS

We tested seven human neuroblastoma cell lines for their sensitivity to imatinib. Cell viability was assessed by trypan blue dye exclusion. Apoptosis was evaluated by nuclear staining, flow cytometry, and western blotting. Protein assays included immunoprecipitation, western blotting, enzyme-linked immunosorbent assays, and immunohistochemistry. mRNA expression was assessed by northern blotting. We used a xenograft model in SCID mice (10 mice per group) to evaluate the effects of imatinib oral therapy (50 or 100 mg/kg every 12 hours for 14 days) on neuroblastoma tumor growth. All statistical tests were two-sided.

RESULTS

All seven neuroblastoma cell lines treated with imatinib displayed concentration-dependent decreases in cell viability, which coincided with an induction of apoptosis, and with ligand-stimulated phosphorylation of c-Kit and PDGFR. The imatinib concentrations that caused 50% inhibition of growth and 50% inhibition of ligand-induced phosphorylation of these receptors were 9-13 micro M and 0.1-0.5 microM, respectively. Expression of VEGF, but not phosphorylation of Flk-1, its receptor, was reduced in neuroblastoma cells treated with imatinib at 10 microM or higher. Mice treated with imatinib at 50 mg/kg or 100 mg/kg had statistically significantly smaller tumors than control mice treated with vehicle (mean tumor volume in mice treated with imatinib at 50 mg/kg = 1546 mm3, in control mice = 2954 mm3; difference = 1408 mm3, 95% confidence interval [CI] = 657 to 2159 mm3; P<.001; mean tumor volume in mice treated with imatinib at 100 mg/kg = 463 mm3; difference = 2491 mm3, 95% CI = 1740 to 3242 mm3; P<.001).

CONCLUSIONS

Imatinib inhibited the growth of neuroblastoma cells in vitro and in vivo. This inhibition was associated with suppression of PDGFR and c-Kit phosphorylation and inhibition of VEGF expression.

In solid cancers, activated fibroblasts acquire the capacity to provide fertile soil for tumor progression. Specifically, cancer-associated fibroblasts (CAFs) establish a strong relationship with cancer cells. This provides advantages to both cell types: whereas cancer cells initiate and sustain CAF activation, CAFs support cancer cell growth, motility and invasion. This results in tumor progression, metastasis and chemoresistance. Numerous studies have detailed the mechanisms involved in fibroblast activation and cancer progression, some of which are reviewed in this article. Cancer cells and CAFs are "partners in crime", and their interaction is supported by inflammation. An understanding of the enemy, the cancer cell population and its "allies" should provide novel opportunities for targeted-drug development. Graphical Abstract Molecular mechanism of fibroblast activation. a Normal fibroblasts are the most common cell type in the extracellular matrix and are responsible for the synthesis of collagens and fibrilar proteins. Under normal conditions, fibroblasts maintain tissue homeostasis and contribute to proper cell communication and function. Fibroblasts can be activated by a diverse set of factors secreted from cancer or immune cells. Not only growth factors such as TGF-β, PDGF, HGF and FGF but also interleukins, metalloproteinases and reactive oxygen species can promote activation. Likewise, transcriptional factors such as NF-κB and HSF-1 play an important role, as do the gene family of metalloproteinase inhibitors, Timp and the NF-κB subunit, p62. Interestingly, fibroblasts themselves can stimulate cancer cells to support activation further. b Once activated, fibroblasts undergo a phenotype switch and become cancer-associated fibroblasts (CAFs) expressing various markers such as α-SMA, FSP1, vimentin and periostatin. c Recently, the LIF/GP130/IL6-R pathway has been identified as a signaling cascade involved in fibroblast activation. Upon LIF stimulation, JAK is phosphorylated and further activates STAT3, a transcriptional factor that is then translocated into the nucleus where it promotes the transcription of genes responsible for cell growth, differentiation, proliferation and apoptosis. Ruxolitinib can inhibit JAK and prevent STAT3 activation. Further on, the maintenance of JAK activation is supported by epigenetical changes and post-translational modifications. Once pSTAT3 is acetylated by histon acetyltransferase, p300, it leads to the loss of expression of SHP-1, which is a negative regulator of the JAK/STAT pathway. Silencing of SHP-1 steers the constitutive activation of JAK and STAT3.

The tumour suppressor protein, PTEN (phosphatase and tensin homolog deleted on chromosome 10), is a phosphatase that can dephosphorylate tyrosine-containing peptides, Shc, focal adhesion kinase and phosphoinositide substrates. In cellular assays, PTEN has been shown to antagonize the PI-3K-dependent activation of protein kinase B (PKB) and to inhibit cell spreading and motility. It is currently unclear, however, whether PTEN accomplishes these effects through its lipid- or protein-phosphatase activity, although strong evidence has demonstrated the importance of the latter for tumour suppression by PTEN. By using a PTEN G129E (Gly(129)->>Glu) mutant that has lost its lipid phosphatase activity, while retaining protein phosphatase activity, we demonstrated a requirement for the lipid phosphatase activity of PTEN in the regulation of PKB activity, cell viability and membrane ruffling. We also made a small C-terminal deletion of PTEN, removing a putative PDZ (PSD95, Dlg and ZO1)-binding motif, with no detectable effect on the phosphatase activity of the protein expressed in HEK293 cells (human embryonic kidney 293 cells) assayed in vitro. Surprisingly, expression of this mutant revealed differential requirements for the C-terminus in the different functional assays. Wild-type and C-terminally deleted PTEN appeared to be equally active in down-regulating PKB activity, but this mutant enzyme had no effect on platelet-derived growth factor (PDGF)-induced membrane ruffling and was only partially active in a cell viability assay. These results stress the importance of the lipid phosphatase activity of PTEN in the regulation of several signalling pathways. They also identify a mutation, similar to mutations that occur in some human tumours, which removes the effect of PTEN on membrane ruffling but not that on PKB.

PURPOSE Dasatinib is an orally available tyrosine kinase inhibitor with low nanomolar activity against SRC family kinases, BCR-ABL, c-KIT, EPHA2, and the PDGF-β receptor. Dasatinib was found to have selective activity in several tumor models in the Pediatric Preclinical Testing Program. PATIENTS AND METHODS A phase I study of dasatinib in pediatric patients with refractory solid tumors or imatinib-refractory, Philadelphia chromosome-positive leukemia was performed. Dose levels of 50, 65, 85, and 110 mg/m²/dose, administered orally twice daily for 28 days, with courses repeated without interruption, were studied. Pharmacokinetic studies were performed with the initial dose.

RESULTS

A total of 39 patients (solid tumors, n = 28; chronic myeloid leukemia [CML], n = 9; acute lymphoblastic leukemia, n = 2) were enrolled. No dose-limiting toxicities (DLTs) were observed at the 50, 65, and 85 mg/m² dose levels. At 110 mg/m², two of six patients experienced DLT including grade 2 diarrhea and headache. In children with leukemia, grade 4 hypokalemia (50 mg/m²), grade 3 diarrhea (85 mg/m²), and grade 2 creatinine elevation (50 mg/m²) were observed. DLT in later courses included pleural effusions, hemangiomatosis, and GI hemorrhage. There were three complete cytogenetic responses, three partial cytogenetic responses, and two partial/minimal cytogenetic responses observed in evaluable patients with CML. CONCLUSION Overall, drug disposition and tolerability of dasatinib were similar to those observed in adult patients.

The c-raf-1 protooncogene encodes a Ser/Thr protein kinase. A mitogen-activated protein kinase-kinase (MAPKK) purified from bovine brain is phosphorylated and activated 4-9-fold in vitro by c-Raf-1 from mitogen-treated cells. c-Raf-1 protein kinase activity, measured by the phosphorylation of brain MAPKK substrate, is detectably activated within 1 min after addition of platelet-derived growth factor (PDGF) to 3T3 cells, increasing more rapidly than the endogenous NIH 3T3 cell MAPKK activity. c-Raf-1 activation is also induced by insulin, phorbol ester, thrombin, and endothelin. PDGF-, epidermal groth factor-, and insulin-stimulated 32P-c-Raf-1 yield very similar, complex tryptic 32P-peptide maps, wherein only 2 of 10 32P-peptides appear entirely de novo after growth factor addition. Mitogen-activated protein kinase/extracellular signal-regulated kinase-2 can phosphorylate c-Raf-1 in vitro on 4-6 tryptic 32P-peptides, all of which comigrate with tryptic 32P-peptides derived from c-Raf-1 labeled in situ. Mitogen-activated protein kinase phosphorylation of c-Raf-1 in vitro, however, does not 1) generate 32P-peptides that comigrate with those that appear de novo after PDGF or insulin treatment in situ; 2) does not convert c-Raf-1 polypeptides to a slower mobility on SDS-polyacrylamide gel electrophoresis as is seen after PDGF or insulin; 3) does not alter c-Raf-1 kinase activity toward MAPKK. Thus, based on overlapping site specificity, Erk-2 is a viable candidate to be among the PDGF-stimulated c-Raf-1 kinases. Although PDGF/insulin-stimulated c-Raf-1 Ser/Thr phosphorylation may be necessary to sustain the active state, a role for mitogen-activated protein kinase/extracellular signal-regulated kinase-2 phosphorylation in the initiation of c-Raf-1 activation is unlikely.

Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice. Am J Physiol Lung Cell Mol Physiol 293: , 2007. First published August 17, 2007; - Mechanical ventilation (MV) with O(2)-rich gas offers life-saving treatment for extremely premature infants with respiratory failure but often leads to neonatal chronic lung disease (CLD), characterized by defective formation of alveoli and blood vessels in the developing lung. We discovered that MV of 2- to 4-day-old mice with 40% O(2) for 8 h, compared with unventilated control pups, reduced lung expression of genes that regulate lung septation and angiogenesis (VEGF-A and its receptor, VEGF-R2; PDGF-A; and tenascin-C). MV with air for 8 h yielded similar results for PDGF-A and tenascin-C but did not alter lung mRNA expression of VEGF or VEGF-R2. MV of 4- to 6-day-old mice with 40% O(2) for 24 h reduced lung protein abundance of VEGF-A, VEGF-R2, PDGF-A, and tenascin-C and resulted in lung structural abnormalities consistent with evolving CLD. After MV with 40% O(2) for 24 h, lung volume was similar to unventilated controls, whereas distal air space size, assessed morphometrically, was greater in lungs of ventilated pups, indicative of impaired septation. Immunostaining for vimentin, which is expressed in myofibroblasts, was reduced in distal lung after 24 h of MV with 40% O(2). These molecular, cellular, and structural changes occurred without detectable lung inflammation as evaluated by histology and assays for proinflammatory cytokines, myeloperoxidase activity, and water content in lung. Thus lengthy MV of newborn mice with O(2)-rich gas reduces lung expression of genes and proteins that are critical for normal lung growth and development. These changes yielded lung structural defects similar to those observed in evolving CLD.

Recent reports have described several cellular phenotypes that appear to be mediated by Endosialin/TEM-1/CD248 (TEM-1), including tubule formation on matrigel, migration and proliferation. It has been shown that siRNA knock-down of TEM-1 in primary human fibroblasts resulted in reduced proliferation. However, the downstream signaling events that mediate TEM-1 function(s) currently remain unknown. In this study, we demonstrate that TEM-1 mediates proliferation of primary human pericytes through a PDGF receptor signaling pathway. Normal pericytes expressing high levels of TEM-1 were able to proliferate, respond to PDGF-BB stimulation by phosphorylating both the PDGF receptor and the MAP kinase ERK-1/2, and induce the expression of the immediate early transcription factor c-Fos. However, when TEM-1 expression was knocked-down, PDGF-BB-induced proliferation, ERK-1/2 phosphorylation, and c-Fos expression were significantly impaired. Thus, our results provide evidence for a TEM-1-dependent signal pathway that controls proliferation of human pericytes and suggest targeting this pathway for future strategies aimed at mitigating tumor angiogenesis.

Candida albicans, the major invasive fungal pathogen of humans, can cause both debilitating mucosal infections and fatal invasive infections. Understanding the complex nature of the host-pathogen interaction in each of these contexts is essential to developing desperately needed therapies to treat fungal infections. RNA-seq enables a systems-level understanding of infection by facilitating comprehensive analysis of transcriptomes from multiple species (e.g., host and pathogen) simultaneously. We used RNA-seq to characterize the transcriptomes of both C. albicans and human endothelial cells or oral epithelial cells during in vitro infection. Network analysis of the differentially expressed genes identified the activation of several signaling pathways that have not previously been associated with the host response to fungal pathogens. Using an siRNA knockdown approach, we demonstrate that two of these pathways-platelet-derived growth factor BB (PDGF BB) and neural precursor-cell-expressed developmentally down-regulated protein 9 (NEDD9)-govern the host-pathogen interaction by regulating the uptake of C. albicans by host cells. Using RNA-seq analysis of a mouse model of hematogenously disseminated candidiasis (HDC) and episodes of vulvovaginal candidiasis (VVC) in humans, we found evidence that many of the same signaling pathways are activated during mucosal (VVC) and/or disseminated (HDC) infections in vivo. Our analyses have uncovered several signaling pathways at the interface between C. albicans and host cells in various contexts of infection, and suggest that PDGF BB and NEDD9 play important roles in this interaction. In addition, these data provide a valuable community resource for better understanding host-fungal pathogen interactions.

BACKGROUND

Sunitinib is a protein tyrosine kinase-inhibitor targeting VEGFR, c-kit and PDGFR. It has been approved for the treatment of metastatic renal-cell carcinoma and gastrointestinal stromal tumors. Although it has been shown to prolong disease-free and overall survival in renal-cell carcinoma patients, only 70% of the treated population receive a clinical benefit (CB) from the treatment. Markers that could predict clinical benefit to sunitinib would be an important aid in monitoring and following their treatment. We assessed the outcome and plasma proangiogenic factors in patients with metastatic renal cell carcinoma (mRCC) treated with sunitinib in our institution.

METHODS

We have treated 42 patients with metastatic clear-cell renal carcinoma with sunitinib. Plasma concentrations of VEGF-A, sVEGFR2 and PDGF were determined by ELISA.

RESULTS

At the time of analysis 39 patients were evaluable for response and 30 patients had obtained a clinical benefit (CB). Median progression-free survival was 268 days (8.93 months) and median overall survival was 487 days (16.23 months). Interestingly, disease stabilization or objective response resulted in comparable overall survival. Most treatment-related adverse events were of mild-to-moderate intensity with one treatment-related death. Plasma sVEGFR2 and PDGF levels had no predictive value. Fold-increase in plasma VEGF was significantly lower in patients that obtained a CB as compared to patients that progressed after two cycles of treatment. Plasma VEGF did not increase in patients with initial CB at the time of progression.

CONCLUSIONS

Sunitinib showed substantial activity in mRCC. Disease stabilization or objective response resulted in comparable overall survival and both outcomes should be considered positive. Fold-increase in plasma VEGF predicts for CB and could be a candidate marker. Progression after initial CB is not associated with elevated plasma VEGF, implying a different mechanism of resistance.

The coupling of the group I metabotropic glutamate receptors, mGlu1a and mGlu5a, to the extracellular signal-regulated protein kinase (ERK) pathway has been studied in Chinese hamster ovary cell-lines where receptor expression is under inducible control. Both mGlu receptors stimulated comparable, robust and agonist concentration-dependent ERK activations in the CHO cell-lines. The mGlu1a receptor-mediated ERK response was almost completely attenuated by pertussis toxin (PTx) pretreatment, whereas the mGlu5a-ERK response, and the phosphoinositide response to activation of either receptor, was PTx-insensitive. mGlu1a and mGlu5a receptor coupling to ERK occurred via mechanisms independent of phosphoinositide 3-kinase activity and intracellular and/or extracellular Ca2+ concentration. While acute treatment with a protein kinase C (PKC) inhibitor did not attenuate agonist-stimulated ERK activation, down-regulation of PKCs by phorbol ester treatment for 24 h did attenuate both mGlu1a and mGlu5a receptor-mediated responses. Further, inhibition of Src non-receptor tyrosine kinase activity by PP1 attenuated the ERK response generated by both receptor subtypes, but only mGlu1a receptor-ERK activation was attenuated by PDGF receptor tyrosine kinase inhibitor AG1296. These findings demonstrate that, although expressed in a common cell background, these closely related mGlu receptors utilize different G proteins to cause ERK activation and may recruit different tyrosine kinases to facilitate this response.

Tenascin-C is an extracellular matrix glycoprotein that is supposed to be a profibrotic molecule in various fibrogenic processes. To elucidate its significance for myocardial fibrosis in the hypertensive heart, we used a mouse model with infusion of angiotensin II and examined results by histology, immunohistochemistry, in situ hybridization, and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Angiotensin II treatment elevated blood pressure and expression of tenascin-C by interstitial fibroblasts in perivascular fibrotic lesions, and angiotensin II infusion caused accumulation of macrophages. It also upregulated expression of collagen Ialpha2; IIIalpha1; and proinflammatory/profibrotic mediators including transforming growth factor beta (TGFbeta), platelet-derived growth factor alpha (PDGF-A), PDGF-B, and PDGF-receptor alpha, but not IL-1beta and PDGF-receptor beta, in the myocardium. Treatment with an aldosterone receptor antagonist, eplerenone, significantly attenuated angiotensin II-induced fibrosis, expression of tenascin-C, and inflammatory changes without affecting the blood pressure level. In vitro, neither eplerenone nor aldosterone exerted any influence on tenascin-C expression of cardiac fibroblasts, whereas angiotensin II, TGF-beta1, and PDGF significantly upregulated expression of tenascin-C. These results suggest that, in the angiotensin II-induced hypertensive mouse heart: (1) tenascin-C may be involved in the progression of cardiac fibrosis and (2) aldosterone may elicit inflammatory reactions in myocardium, which might, in turn, induce tenascin-C synthesis of fibroblasts through at least 2 pathways mediated by TGF-beta and PDGF-A-B/PDGF-receptor alpha.

Migration and proliferation of smooth muscle cells (SMCs) are key events during neointimal formation in pathological conditions of vessels. Tenascin-C (TNC) is upregulated in the developing neointima of lesions. We evaluated the effects of TNC on responses of SMCs against platelet-derived growth factor (PDGF) stimulation. TNC coated on substrate promoted PDGF-BB-induced proliferation and migration of rat SMC cell line A10 in BrdU incorporation and transwell assays, respectively. Immunoblotting showed that TNC substrate enhanced autophosphorylation of PDGFR-β after PDGF-BB stimulation. Integrin αvβ3 is known to be a receptor for TNC in SMCs. In immunofluorescence and immunoblot of integrin αv subunit, clustering of αv-positive focal adhesions and upregulated αv expression were observed in the cells on TNC substrate. Immunoprecipitation using anti-integrin αvβ3 antibody demonstrated that PDGFR-β and integrin αvβ3 were co-precipitated and that the relative amount of PDGFR-β after the stimulation was increased by TNC treatment. TNC also promoted phosphorylation of focal adhesion kinase (FAK) at tyrosine (Y) 397 and Y925. The phosphorylated FAK was localized at focal adhesions in immunofluorescence. Phosphorylated SRC at Y418 was also seen at focal adhesions. Immunoprecipitation with αv antibody showed increased SRC association with the integrin signaling complex in the cells on TNC after PDGF treatment. In the cells on TNC substrate, crosstalk signaling between integrin αvβ3 and PDGFR-β could be amplified by SRC and FAK recruited to focal adhesions, followed by enhanced proliferation and migration of A10 cells by PDGF-BB.

Tyrosine kinases regulate a broad variety of physiological cell processes, including metabolism, growth, differentiation and apoptosis. Abnormal tyrosine kinase activity disturbs the physiological cell homeostasis and can lead to cancer, vascular disease, and fibrosis. In regard to fibrosis, different tyrosine kinases have been identified as determinants of disease progression and potential targets for anti-fibrotic therapies. This includes both receptor tyrosine kinases (e.g., PDGF receptor, VEGF receptor, EGF receptor, and JAK kinases) as well as non-receptor tyrosine kinases (e.g., c-Abl, c-Kit, and Src kinases). Given their central role in the pathogenesis of fibrosis, researchers of our field study the anti-fibrotic effects of monoclonal antibodies or small-molecule inhibitors to block the aberrant tyrosine kinase activity and treat fibrosis in preclinical models of various fibrotic diseases (e.g., idiopathic pulmonary fibrosis, renal fibrosis, liver fibrosis, and dermal fibrosis). The results of these studies were promising and prompted clinical trials with different compounds in fibrotic diseases. So far, results from studies with intedanib in idiopathic pulmonary fibrosis and imatinib in idiopathic pulmonary fibrosis and systemic sclerosis have been reported. Although none of these studies reported a positive primary outcome, promising trends in anti-fibrotic efficacy awaken our hopes for a new class of effective anti-fibrotic targeted therapies. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

Airway remodeling, as manifested by an increase in airway smooth muscle mass, mucous gland hyperplasia, and subepithelial fibrosis, contributes to the airway hyperresponsiveness and fixed obstruction seen in some asthmatic patients. Here we investigated whether the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway contributes to platelet-derived growth factor (PDGF)-stimulated mitogenesis of human airway smooth muscle cells (HASMC). PDGF treatment of quiescent HASMC resulted in the rapid tyrosine phosphorylation and DNA binding of STAT1 and STAT3. This phosphorylation was blocked by inhibition of Src and JAK2 kinases. In addition, STAT activation by PDGF was found to be redox dependent. Moreover, PDGF-induced thymidine uptake was completely blocked by pretreatment of HASMC with the STAT kinase inhibitors AG-490, SU-6656, and PP2. Interestingly, the JAK pathway was required for HASMC mitogenesis independently of mitogen-activated protein kinase activation. Inhibition of the Src and JAK kinases blocked PDGF-stimulated gene expression of the STAT target genes cyclin D1 and c-myc. These results indicate that the JAK-STAT pathway contributes to PDGF-induced mitogenesis, and thus this pathway may be important in the airway remodeling seen in some asthmatic patients.

Two homobifunctional cross-linking reagents have been used to cross-link 125I-platelet-derived growth factor (PDGF) to a cell surface component with an approximate Mr = 164,000 that has many characteristics of a specific PDGF receptor. Excess unlabeled PDGF competed for labeling of this component, while high concentrations of fibroblast growth factor, insulin, epidermal growth factor, low density lipoprotein or acetylated low density lipoprotein had no effect. Preincubation of cells with 125I-PDGF at 37 degrees C reduced specific 125I-PDGF binding (down regulation) and produced a parallel decrease in the amount of the 164,000-dalton receptor available for labeling. The 164,000-dalton component contains at least some protein that is accessible to trypsin in the extracellular medium. A complex of comparable Mr is seen on all PDGF-responsive cell types examined, but not on a nonresponsive cell type. 125I-PDGF does not become covalently cross-linked to this component in the absence of a cross-linking reagent.