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 PDD(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.

The wild-type and two mitogenic-defective mutants of the type beta receptor for platelet-derived growth factor (PDGF) were expressed in Chinese hamster ovary cells. In the first mutant, delta Ki, 82 of 104 amino acids in the kinase insert region were deleted. This mutant was recently reported to be defective in mediating DNA synthesis. In the second mutant, Y825F, tyrosine 825 was converted to phenylalanine by a point mutation. We report here that this mutant is also defective in mediating PDGF-stimulated DNA synthesis. Both mutants were capable of eliciting many of the early responses to PDGF, including receptor autophosphorylation. However, neither mutant was capable of undergoing PDGF-stimulated change in receptor conformation or of phosphorylating exogenous substrate in an in vitro assay. These data suggest that changes in receptor conformation and efficient utilization of specific tyrosine kinase substrates are important for the stimulation of cell proliferation of PDGF and that phosphorylation of tyrosine 825 may be involved in signal transduction.

OBJECTIVE

Unlike platelet-derived growth factor-B (PDGF-B), the role of PDGF-D in tumor progression or treatment is largely unknown. To this end, we determined the role of PDGF-D in breast cancer progression, metastasis, and response to chemotherapy.

METHODS

We first examined PDGF-D expression in human breast carcinomas by immunohistochemical (IHC) staining. To mimic high PDGF-D expressing tumors, we stably transfected the breast cancer cell lines MDA-MB-231 and 4T1 with pdgf-d cDNA, and implanted these tumor cells orthtopically into nude mice. We monitored tumor growth by caliper measurement and bioluminescence imaging. We also used short hairpin RNA interference (shRNAi) and imatinib to block PDGF-D/PDGFRβ signaling. Finally, we studied the effect of PDGF-D on doxorubicin delivery and efficacy.

RESULTS

Human breast cancers express high levels of PDGF-D. Overexpression of PDGF-D promoted tumor growth and lymph node metastasis through increased proliferation, decreased apoptosis, and induction of CXCR4 expression. Blockade of CXCR4 signaling abolished PDGF-D-induced lymph node metastasis. Furthermore, overexpression of PDGF-D increased perivascular cell coverage and normalized tumor blood vessels. As a result, PDGF-D overexpression facilitated tissue penetration of doxorubicin and enhanced its treatment efficacy.

CONCLUSIONS

PDGF-D is highly expressed in human breast cancer and facilitates tumor growth and lymph node metastasis, making it a potential target in breast cancer. At the same time, PDGF-D increases drug delivery and hence improves the efficacy of chemotherapy through vessel normalization. Therefore, judicious use of PDGF-D/PDGFRβ blockers would be necessary to minimize the adverse effects on concomitantly administered cytotoxic therapies.

In this study, we investigated the functional role of the transcription factor AP-1 in hypoxia-induced expression of the vascular endothelial growth factor (VEGF) by using dexamethasone as an inhibitor of AP-1 activity. Phorbol ester and platelet-derived growth factor (PDGF) cause an increase in VEGF mRNA expression, which is strongly suppressed in the presence of dexamethasone, whereas hypoxia-induced VEGF expression is not inhibited by dexamethasone. Studies using a VEGF promoter luciferase construct show that the phorbol ester and PDGF-induced VEGF expression is mediated at least in part by transcriptional activation of the VEGF promoter, whereas no transcriptional activation is seen under hypoxic conditions. In contrast, hypoxia leads to an increase in VEGF mRNA stability, as confirmed by experiments using actinomycin D as an inhibitor of transcription. These results indicate that hypoxia-induced VEGF expression is independent of AP-1 mediated transcription.

This study provides new perspectives of the unique aspects of platelet-derived growth factor beta-receptor (PDGFR-beta) signaling and biological responses through the establishment of a mutant mouse strain in which two loxP sequences were inserted into the introns of PDGFR-beta genome sequences. Isolation of skin fibroblasts from the mutant mice and Cre recombinase transfection in vitro induced PDGFR-beta gene deletion (PDGFR-betaDelta/Delta). The resultant depletion of the PDGFR-beta protein significantly attenuated platelet-derived growth factor (PDGF)-BB-induced cell migration, proliferation, and protection from H2O2-induced apoptosis of the cultured PDGFR-betaDelta/Delta dermal fibroblasts. PDGF-AA and fetal bovine serum were mitogenic and anti-apoptotic but were unable to induce the migration in PDGFR-beta Delta/Delta fibroblasts. Concerning the PDGF signaling, PDGF-BB-induced phosphorylation of Akt, ERK1/2, and JNK, but not p38, decreased in PDGFR-betaDelta/Delta fibroblasts, but PDGF-AA-induced signaling was not altered. Overexpression of the phospholipid phosphatases, SHIP2 and/or PTEN, inhibited PDGF-BB-induced phosphorylation of Akt and ERK1/2 in PDGFR-betaDelta/Delta fibroblasts but did not affect that of JNK and p38. These results indicate that disruption of distinct PDGFR-beta signaling pathways in PDGFR-betaDelta/Delta dermal fibroblasts impaired their proliferation and survival, but completely inhibits migratory response, and that PDGF-BB-induced phosphorylation of Akt and ERK1/2 possibly mediated by PDGFR-alpha is regulated, at least in part, by the lipid phosphatases SHIP2 and/or PTEN. Thus, the PDGFR-beta function on dermal fibroblasts appears to be critical in PDGF-BB action for skin wound healing and is clearly distinctive from that of PDGFR-alpha in the ligand-induced biological responses and the underlying properties of cellular signaling.

An SH2 domain originally termed SH2-B had been identified as a direct cellular binding target of a number of mostly mitogenic receptors. The complete cellular protein, termed PSM, and respective sequence variants share additional Pro-rich and PH regions, as well as similarities with APS and Lnk. A role of these mediators has been implicated in signaling pathways found downstream of growth hormone receptor and receptor tyrosine kinases, including the insulin, insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), nerve growth factor, hepatocyte growth factor, and fibroblast growth factor receptors. As a result of this report a total of four PSM/SH2-B sequence variants termed alpha, beta, gamma, and delta have now been identified in the mouse and have been compared with the available rat and human sequences. Variant differences are based on alternative splicing and define distinct last exons 7, 8, and 9 that result in reading frameshifts and unique carboxyl-terminal amino acid sequences. Variant sequences have been identified from cDNA libraries and directly by reverse transcription-polymerase chain reaction. Sequence analysis predicts four distinctly sized protein products that have been demonstrated after cDNA expression. All were found phosphorylated on tyrosine specifically in response to IGF-I and PDGF stimulation. cDNA expression of the four variants caused variant-dependent levels of stimulation of IGF-I- and PDGF-induced mitogenesis. The most pronounced increase in mitogenesis was consistently observed for the gamma variant followed by delta, alpha, and beta with decreasing responses. In contrast, the mitogenic response to epidermal growth factor consistently remained unaffected. The variants are expressed in most mouse tissues, typically, most strongly in pairs of alpha and delta or beta and gamma. Our findings implicate differential roles of the PSM/SH2-B splice variants in specific mitogenic signaling pathways.

Previous investigators reported that epiretinal membranes isolated from patients with proliferative vitreoretinopathy (PVR) express various platelet-derived growth factor (PDGF) family members and PDGF receptors (PDGFRs) (Cui, J.Z., Chiu, A., Maberley, D., Ma, P., Samad, A., Matsubara, J.A., 2007. Stage specificity of novel growth factor expression during development of proliferative vitreoretinopathy. Eye 21, 200-208; Robbins, S.G., Mixon, R.N., Wilson, D.J., Hart, C.E., Robertson, J.E., Westra, I., Planck, S.R., Rosenbaum, J.T., 1994. Platelet-derived growth factor ligands and receptors immunolocalized in proliferative retinal diseases. Invest. Ophthalmol. Vis. Sci. 35(10), 3649-3663). Co-expression of ligand and receptor raises the possibility of an autocrine loop, which could be of importance in the pathogenesis of PVR. To begin to address this issue we determined whether the PDGFRs in epiretinal membranes isolated from PVR patient donors were activated. Indeed, immunohistochemical staining (using pan- and phospho-PDGFR antibodies) revealed that both PDGFR subunits were activated. Quantification of these data demonstrated that a greater percentage of cells expressed the PDGFR alpha subunit as compared with the beta subunit (44 +/- 13% versus 32 +/- 6.5%). Staining with phospho-PDGFR antibodies indicated that 36 +/- 10% of the PDGFR alpha subunits were activated, whereas only 16 +/- 5.5% of the PDGFR beta subunits were activated. Thus, a 2.25 fold greater percentage of the PDGFR alpha subunits was activated. Co-staining with diagnostic cell-type antibodies indicated that both retinal pigment epithelial and glial cells expressed activated PDGFR alpha subunits. These findings support the recent discovery that PDGF-C is the major vitreal isoform because PDGF-C is 3 times more likely to activate a PDGFR alpha subunit as compared with a PDGFR beta subunit. We conclude that PDGFRs are activated in epiretinal membranes of patients with PVR, and that the profile of active PDGFR subunits functionally supports the idea that PDGF-C is the predominant PDGF isoform present in the vitreous of patients with PVR. These findings identify PDGF-A, -AB and C as the best therapeutic targets within the PDGF family.

Several lines of evidence suggest that imatinib may affect skeletal tissue. We show that inhibition by imatinib of PDGFR signaling in osteoblasts activates osteoblast differentiation and inhibits osteoblast proliferation and that imatinib inhibits osteoclastogenesis by both stromal cell-dependent and direct effects on osteoclast precursors.

BACKGROUND

Imatinib mesylate, an orally active inhibitor of the c-abl, c-kit, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, is in clinical use for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal cell tumors. Interruption of both c-kit and c-abl signaling in mice induces osteopenia, suggesting that imatinib might have adverse effects on the skeleton. However, biochemical markers of bone formation increase in patients with CML starting imatinib therapy, whereas bone resorption is unchanged, despite secondary hyperparathyroidism. We assessed the actions of imatinib on bone cells in vitro to study the cellular and molecular mechanism(s) underlying the skeletal effects we observed in imatinib-treated patients.

METHODS

Osteoblast differentiation was assessed using a mineralization assay, proliferation by [(3)H]thymidine incorporation, and apoptosis by a TUNEL assay. Osteoclastogenesis was assessed using murine bone marrow cultures and RAW 264.7 cells. RT and multiplex PCR were performed on RNA prepared from human bone marrow samples, osteoblastic cells, and murine bone marrow cultures. Osteoprotegerin was measured by ELISA.

RESULTS

The molecular targets of imatinib are expressed in bone cells. In vitro, imatinib increases osteoblast differentiation and prevents PDGF-induced inhibition of this process. Imatinib inhibits proliferation of osteoblast-like cells induced by serum and PDGF. In murine bone marrow cultures, imatinib inhibits osteoclastogenesis stimulated by 1,25-dihydroxyvitamin D(3) and partially inhibits osteoclastogenesis induced by RANKL and macrophage-colony stimulating factor. Imatinib partially inhibited osteoclastogenesis in RANKL-stimulated RAW-264.7 cells. Treatment with imatinib increases the expression of osteoprotegerin in bone marrow from patients with CML and osteoblastic cells.

CONCLUSIONS

Taken together with recent in vivo data, these results suggest a role for the molecular targets of imatinib in bone cell function, that inhibition by imatinib of PDGFR signaling in osteoblasts activates bone formation, and that the antiresorptive actions of imatinib are mediated by both stromal cell-dependent and direct effects on osteoclast precursors.

Platelet-derived growth factor receptors (PDGFR) α and β have been suggested as potential targets for treatment of rhabdomyosarcoma, the most common soft tissue sarcoma in children. This study identifies biologic activities linked to PDGF signaling in rhabdomyosarcoma models and human sample collections. Analysis of gene expression profiles of 101 primary human rhabdomyosarcomas revealed elevated PDGF-C and -D expression in all subtypes, with PDGF-D as the solely overexpressed PDGFRβ ligand. By immunohistochemistry, PDGF-CC, PDGF-DD, and PDGFRα were found in tumor cells, whereas PDGFRβ was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFRα signaling associated with genes involved in the reactivation of developmental programs, PDGF-DD/PDGFRβ signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFRβ in vascular stroma and the alveolar subtype and with presence of metastases. Functional validation of our findings was carried out in molecularly distinct model systems, where therapeutic targeting reduced tumor burden in a PDGFR-dependent manner with effects on cell proliferation, vessel density, and macrophage infiltration. The PDGFR-selective inhibitor CP-673,451 regulated cell proliferation through mechanisms involving reduced phosphorylation of GSK-3α and GSK-3β. Additional tissue culture studies showed a PDGFR-dependent regulation of rhabdosphere formation/cancer cell stemness, differentiation, senescence, and apoptosis. In summary, the study shows a clinically relevant distinction in PDGF signaling in human rhabdomyosarcoma and also suggests continued exploration of the influence of stromal PDGFRs on sarcoma progression.

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K(d)) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K(d) of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K(d).

BACKGROUND

Cerebrovascular complications involving endothelial dysfunction at the blood-brain barrier (BBB) are central to the pathogenesis of diabetes-related CNS disorders. However, clinical and experimental studies have reported contrasting evidence in relation to the effects of hyperglycemia on BBB permeability and function. Similarly the effect of hypoglycemia on BBB integrity is not well understood. Therefore, we assessed the differential impact of hypo and hyperglycemic conditions on BBB integrity and endothelial function in vitro using hCMEC/<em>D</em>3, a well characterized human brain microvascular endothelial cell line.

METHODS

Parallel monolayers of hCMEC/DD-glucose, respectively. Following 3-24h exposure, the expression and distribution of BBB tight junction (ZO-1 and claudin-5) adherence junction (VE-cadherin) proteins, and glucose transporters as well as inflammatory (VCAM-1) and oxidative stress (Nrf-2) markers were analyzed by immunofluorescence and western blotting. Endothelial release of growth factors and pro-inflammatory cytokines were determined by ELISA. Further, the impact of altered glycemia on BBB permeability was assessed in hCMEC/DDa).

RESULTS

Compared to controls, exposure to hypoglycemia (3 and 24h) down-regulated the expression of claudin-5 and disrupted the ZO-1 localization at cell-cell contacts, while hyperglycemia marginally reduced claudin-5 expression without affecting ZO-1 distribution. Permeability to dextrans (4-10 kDa) and VEGF release at 24h were significantly increased by hypo- and hyperglycemia, although 70 kDa dextran permeability was increased only under hypoglycemic conditions. The expression of SGLT-1 was up-regulated at 24h hypoglycemic exposure while only a modest increase of GLUT-1 expression was observed. In addition, the expression of Nrf-2 and release of interleukin-6 and PDGF-BB, were down-regulated by hypoglycemia (but not hyperglycemia), while both conditions induced a marginal and transient increase in VCAM-1 expression from 3 to 24h, including a significant increase in VE-cadherin expression at 3 h following hyperglycemia.

CONCLUSIONS

In summary, our findings demonstrate a potential impairment of BBB integrity and function by hypo or hyperglycemia, through altered expression/distribution of TJ proteins and nutrient transporters. In addition, hypoglycemic exposure severely affects the expression of oxidative and inflammatory stress markers of BBB endothelium.

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.

The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.

Vascular smooth muscle cell (VSMC) activation promotes a synthetic phenotype that underlies many vessel growth disorders. In this regard it has been suggested that the metabolic sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) has significant antigrowth and antimetastatic properties and may serve as a viable therapeutic target. In the current study we hypothesized that AMPK reduces neointima formation following balloon injury and that this occurs through reduction in VSMC proliferation and migration. Data reveal that local or systemic dosing with the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) significantly increased AMPK activity in vivo and inhibited neointima formation in rat carotid arteries 2 wk after injury. In primary VSMCs, AICAR inhibited migration and induced cytostatic growth arrest through increased protein phosphatase 2A-mediated inhibition of mitosis-promoting cyclin B. AICAR also significantly enhanced AMPK-specific T278 phosphorylation of the actin anticapping vasodilator-activated serum phosphoprotein, increased G- to F-actin ratios and stress fiber formation, and abrogated PDGF-stimulated S397 autophosphorylation of focal adhesion kinase, promigratory cytoplasmic accumulation of paxillin, and extracellular matrix proteolysis by matrix metalloproteinase-9. Together, these results provide compelling evidence that AMPK serves to inhibit vascular smooth muscle migration and proliferation through regulation of cytoskeletal/focal adhesion/ECM stability, increasing our knowledge of this important metabolic regulator and providing support for its continued investigation in the treatment of vascular growth disorders.

Platelet-derived growth factor-D (PDGF-D) has been linked with several human malignancies; however, its role in breast cancer progression is not known. We found that PDGF-D expressing breast cancer cell lines MDA-MB-231 and SUM-149 are more invasive compared to cell lines with little or no expression of PDGF-D such as MDA-MB-468 and MCF-7 cells. Over-expression of PDGF-D in PDGF-D low expressing MDA-MB-468 and MCF-7 cells by cDNA transfection showed increased cell proliferation while silencing the expression of PDGF-D by siRNA in PDGF-D high expressing MDA-MB-231 and SUM-149 cells showed decreased cell proliferation and increased apoptosis. Moreover, PDGF-D over-expression was positively correlated with the expression of Notch-1 and Jagged-1, and the expression of mesenchymal markers (Vimentin and ZEB-2) with concomitant decreased expression of epithelial marker E-cadherin. Since NF-κB activation plays a crucial role in Notch signaling as well as in epithelial-mesenchymal transition and tumor aggressiveness, we determined the DNA binding activity of NF-κB and our findings are consistent showing that PDGF-D over-expression led to increased DNA binding activity of NF-κB while it was found to be decreased by inactivation of PDGF-D. These results were also consistent with the expression and activity of MMP-9 and VEGF, as well as invasive characteristics. Further, forced expression of Notch-1/Jagged-1 by cDNA transfection de-repressed the effects of PDGF-D silencing on NF-κB activity and invasion. From these results, we conclude that PDGF-D plays an important role in breast tumor aggressiveness and this process is mechanistically linked with the activation of Notch and NF-κB signaling.

We have investigated the mechanisms involved in H2O2-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Calpha (PKCalpha), and a 62-kDa protein in rat brain PLDDPDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H2O2 generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCalpha. Stimulation by H2O2 showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLDD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H2O2-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H2O2 was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate also abolished H2O2-stimulated PLD activity. H2O2 or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLDD activation. Reduction of intracellular H2O2 levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLDD activation, suggesting the potential role of intracellular H2O2 in H2O2-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H2O2-induced PLD activation in Swiss 3T3 cells.

Hepatocellular carcinoma (HCC) is one of the common malignances in the world and has high mortality in part due to development of acquired drug resistance. Therefore, it is urgent to investigate the molecular mechanism of drug resistance in HCC. To explore the underlying mechanism of drug resistance in HCC, we developed gemcitabine-resistant (GR) HCC cells. We used multiple methods to achieve our goal including RT-PCR, Western blotting analysis, transfection, Wound-healing assay, migration and invasion assay. We observed that gemcitabine-resistant cells acquired epithelial-mesenchymal transition (EMT) phenotype. Moreover, we found that PDGF-D is highly expressed in GR cells. Furthermore, down-regulation of PDGF-D in GR cells led to partial reversal of the EMT phenotype. Our findings demonstrated that targeting PDGF-D could be a novel strategy to overcome gemcitabine resistance in HCC.

Human platelet-derived growth factor (PDGF) is a potent mitogenic polypeptide which is believed to be a heterodimer of A- and B-chains stabilized by interchain disulphide bonds. The B-chain of PDGF is encoded by the c-sis gene, the normal cellular homologue of the transforming gene of the simian sarcoma virus (SSV). cDNA clones of the B-chain from both normal and transformed cells have mutually consistent DNA sequences. Recently, an A-chain cDNA clone (D-1) was isolated from a transformed human glial cell cDNA library. We report the complete sequence of an A-chain cDNA clone (BT-1) isolated from a normal human umbilical vein endothelial (HUVE) cell cDNA library. BT-1 differs from the sequence of the D-1 clone by a 69 base pair deletion containing the predicted carboxy terminus of the protein. The mRNA levels of the A- and B-chains of PDGF in HUVE cells were analysed and shown to respond differently to the endothelial cell growth factor (ECGF).

Lysophosphatidate (LPA) mediates multiple cellular responses via heterotrimeric G protein coupled LPA-1, LPA-2, and LPA-3 receptors. Many G protein-coupled receptors stimulate ERK following tyrosine phosphorylation of growth factor receptors; however, the mechanism(s) of transactivation of receptor tyrosine kinases are not well defined. Here, we provide evidence for the involvement of phospholipase <em>D</em> (PL<em>D</em>) in LPA-mediated transactivation of platelet-derived growth factor receptor-beta (<em>PDGF</em>-R beta). In primary cultures of human bronchial epithelial cells (HBEpCs), LPA stimulated tyrosine phosphorylation of <em>PDGF</em>-R beta and threonine/tyrosine phosphorylation of ERK1/2. The LPA-mediated activation of ERK and tyrosine phosphorylation of <em>PDGF</em>-R beta was attenuated by tyrphostin AG 1296, an inhibitor of <em>PDGF</em>-R kinase, suggesting transactivation of <em>PDGF</em>-R by LPA. Furthermore, LPA-, but not <em>PDGF</em> beta-chain homodimer-induced tyrosine phosphorylation of <em>PDGF</em>-R beta was partially blocked by pertussis toxin, indicating coupling of LPA-R(s) to Gi. Exposure of HBEpCs to LPA activated PL<em>D</em>. Butan-1-ol, which acts as an acceptor of phosphatidate generated by the PL<em>D</em> pathway, blocked LPA-mediated transactivation of <em>PDGF</em>-R beta. This effect was not seen with butan-3-ol, suggesting PL<em>D</em> involvement. The role of PL<em>D</em>1 and PL<em>D</em>2 in the <em>PDGF</em>-R beta transactivation by LPA was investigated by infection of cells with adenoviral constructs of wild type and catalytically inactive mutants of PL<em>D</em>. LPA activated both PL<em>D</em>1 and PL<em>D</em>2 in HBEpCs; however, infection of cells with c<em>D</em>NA for wild type PL<em>D</em>2, but not PL<em>D</em>1, increased the tyrosine phosphorylation of <em>PDGF</em>-R beta in response to LPA. Also, the LPA-mediated tyrosine phosphorylation of <em>PDGF</em>-R beta was attenuated by the catalytically inactive mutant mPL<em>D</em>2-K758R. Infection of HBEpCs with adenoviral constructs of wild type hPL<em>D</em>1, mPL<em>D</em>2, and the inactive mutants of hPL<em>D</em>1 and mPL<em>D</em>2 resulted in association of PL<em>D</em>2 wild type and inactive mutant proteins with the <em>PDGF</em>-R beta compared with PL<em>D</em>1. These results show for the first time that transactivation of <em>PDGF</em>-R beta by LPA in HBEpCs is regulated by PL<em>D</em>2.

In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.

The expression and localization of PDGF beta receptors and PDGF-AB/BB in human healing wounds was evaluated by immunohistochemical techniques and in situ hybridization. Expression of PDGF beta receptor protein and PDGF-AB/BB were analyzed in wound margin biopsies using the PDGFR-B2 and PDGF 007 antibodies. PDGF beta receptor expression was minor in normal skin. An increased expression of PDGF beta receptor protein was prominent in vessels in the proliferating tissue zone in wounds as early as 1 d after surgery and was apparent < or = 4 wk after surgery. There was also a concordant increase in PDGF beta receptor mRNA detected by in situ hybridization. PDGF-AB/BB was present in healing wounds as well as in normal skin. In normal skin, expression of PDGF-AB/BB was confined to peripheral nerve fibers and to solitary cells of the epidermis and of the superficial dermis. In wounds, infiltrating mononuclear cells also stained for PDGF-AB/BB. To identify cell types expressing PDGF AB/BB and PDGF beta receptors, respectively, we performed double immunofluorescence stainings. PDGF beta receptors were expressed by vascular smooth muscle cells and cells in capillary walls; the receptor protein could not be detected in neurofilament containing structures, T lymphocytes, or CD68 expressing macrophages. PDGF-AB/BB colocalized with neurofilaments, it was present in Langerhans cells of the epidermis and in HLA-DR positive cells located in the epidermal/dermal junction area. Of the macrophages infiltrating the wound, 43 +/- 18% stained positively for PDGF AB/BB. Since PDGF-AB/BB and PDGF beta receptors are expressed in the healing wound, two essential prerequisites for a role of PDGF in wound healing are fulfilled.

The effects of 5 different growth factors [EGF, PDGF(bb), TGF-alpha, bFGF and IL-2] were studied on tumour spheroids obtained from 5 different human glioma cell lines (U-251MG, D-263MG, D-37MG, D-54MG, GaMG). The expression of EGF and PDGF receptors as well as the endogenous production of TGF-alpha and PDGF were studied by Northern blot analyses. After growth-factor-exposure, tumour spheroid volume growth, and directional cell migration from the spheroids were studied. In addition, tumour-cell invasion was studied in vitro, where foetal rat-brain aggregates were used as a target for the tumour cells. In all the assays a common stimulator for most of the cell lines was EGF. The other growth factors had a more heterogeneous stimulatory effect. Tumour-cell invasion, cell growth and cell migration are biological properties which are not necessarily related to each other. This may explain why the tumours often responded differently to the growth factors in the various assay systems. Two of the cell lines studied were non-invasive (U-251MG, D-263MG). It is shown that these were stimulated both in the directional migration assay and in the spheroid-volume-growth assay. However, their non-invasive behaviour was not influenced by the growth factors studied.

Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule) form a heterodimeric complex that associates with endosomal membranes and is tyrosine-phosphorylated in response to a variety of growth factors including EGF (epidermal growth factor), HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). Phosphorylation of the Hrs-STAM complex requires receptor endocytosis. We show that an intact UIM (ubiquitin interaction motif) within Hrs is a conserved requirement for Hrs phosphorylation downstream of both EGF and HGF stimulations. Consistent with this, expression of a dominant-negative form of the E3 ubiquitin ligase, c-Cbl, inhibits EGF- and HGF-dependent Hrs phosphorylation. Despite this conservation, kinase inhibitor profiles using PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and SU6656 indicate that distinct non-receptor tyrosine kinases couple EGF, HGF and PDGF stimulation with the tyrosine phosphorylation of the Hrs-STAM complex. Crucially, analysis with phospho-specific antibodies indicates that these kinases generate a signal-specific, combinatorial phosphorylation profile of the Hrs-STAM complex, with the potential of diversifying tyrosine kinase receptor signalling through a common element.

BACKGROUND

Atherosclerosis and vascular calcifications are common causes of morbidity and mortality in maintenance haemodialysis patients. In addition to the well-known traditional risk factors, uraemia-specific factors appear to enhance dramatically the progression of the pathological processes involved. The aim of the present study was to evaluate the degree of atherosclerosis and vascular calcifications in chronic haemodialysis patients using non-invasive imaging methods, and to identify potentially involved factors.

METHODS

The study included 73 patients (36 females, 37 males), aged 25-75 years, who were on haemodialysis treatment for 12-275 months (mean dialysis vintage 73.8 months). We assessed the following circulating parameters: calcium (Ca), phosphorus, 'intact' parathyroid hormone (iPTH), 25OH vitamin D, lipids, oxidized LDL (ox-LDL), Lp(a), homocysteine, leptin, IL-1-beta, IL-6, CRP, TGF-beta, TNF-alpha, (PDGF), advanced oxidation protein products (AOPP) and myeloperoxidase activity (MPO). Coronary artery calcification score (CACS) was assessed using multi-row spiral CT (MSCT). Intima-media thickness index of the common carotid artery (CCA-IMT) and presence of cervical artery atherosclerotic plaques were evaluated by ultrasonography.

RESULTS

Coronary artery calcifications were observed in 79.5% of the patients, with CACS ranging from 0 to 4987. In univariate analysis, a positive correlation was observed between CACS and age, BMI, iPTH, CRP, IL-6 and CCA-IMT, whereas an inverse correlation existed with 25OH vitamin D, TGF-beta and PDGF. CCA-IMT ranged from 0.4 to 1.1 mm. It was positively correlated, in univariate analysis, with age, CACS, CRP and Il-6, and negatively with 25OH vitamin D, TGF-beta and PDGF. Only CACS remained as independent predictive factor of CCA-IMT in multivariate analysis. Atherosclerotic plaques were found in the carotid arteries of 53 patients (72%). The number of plaques was positively correlated with age, CACS, phosphorus, MPO, CRP and IL-6, and inversely with 25OH vitamin D in univariate analysis. In multivariate regression analysis, only age and CACS remained as independent variables.

CONCLUSIONS

In addition to classic risk factors, the degree of atherosclerosis and vascular calcification in our dialysis patient population were associated with several factors that are frequently abnormal in advanced chronic renal failure, but except age, all of them were interdependent. Notably, as in the general population, CACS was an independent predictor of the degree of atherosclerosis in haemodialysis patients.