We have investigated synthesis of 3-phosphorylated inositol lipids in growth factor-stimulated Swiss 3T3 cells. Those growth factors tested which act via tyrosine kinase-containing receptors (platelet-derived growth factor (PDGF), insulin growth factor I (IGF-I), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF)) caused the rapid synthesis of [32P]PtdIns(3,4)P2 and [32P]PtdIns(3,4,5)P3 (PtdIns is phosphatidylinositol) in [32P]P(i)-prelabeled cells and the appearance of an inositol lipid 3-OH kinase in antiphosphotyrosine immunoprecipates. In contrast, those growth factors tested which act via G-protein-coupled receptors (bombesin, vasopressin, prostaglandin E1) were unable to stimulate either of the above responses. Furthermore, while PDGF was able to increase the formation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in streptolysin-permeabilized cells, guanosine 5'-3-(thio)triphosphate and guanyl-5'-yl imidodiphosphate were not. These results suggest that Swiss 3T3 cells possess the machinery for tyrosine kinase but not G-protein-mediated activation of PtdIns(4,5)P2 3-OH kinase; a situation which is the inverse to that recently described for human neutrophils. The tyrosine kinase-containing receptors differed markedly in their relative abilities to elevate the levels of [32P] PtdIns(3,4,5)P3 (ranked in the order PDGF greater than or equal to IGF-I greater than EGF greater than bFGF), [32P]Ptd-OH (PDGF greater than EGF greater than bFGF; undetectable for IGF-I), and [32P]PtdIns4P (EGF greater than bFGF greater than PDGF; undetectable for IGF-I) in [32P]P(i)-prelabeled cells. These differences are epitomized by IGF-I, which was the joint most powerful stimulus for [32P] PtdIns(3,4,5)P3 formation, but was unable to stimulate a measurable accumulation of [32P]Ptd-OH (and hence, by deduction, was unable to stimulate phospholipase C). These results indicate that there is a differential ability among the tyrosine kinase-containing receptors present in a single cell to recruit phospholipase C and PtdIns(4,5)P2 3-OH kinase into their signalling complexes and further emphasizes the notion that the rapid synthesis of PtdIns(3,4,5)P3 may be a signalling event.

Imatinib mesylate (imatinib) is highly effective in the treatment of chronic myeloid leukemia (CML) but is less effective in eliminating CML stem cells. We investigated whether SKI-606, a potent Bcr-Abl and Src kinase inhibitor without anti-PDGF or c-Kit activity, could effectively target primitive CML progenitors. CML and normal progenitors were cultured with SKI-606 or imatinib. SKI-606 effectively inhibited Bcr-Abl kinase activity in CML CD34(+) cells and inhibited Src phosphorylation more potently than imatinib. However, SKI-606 and imatinib resulted in similar suppression of CML primitive and committed progenitor proliferation and growth in CFC and LTC-IC assays. Exposure to either agent alone or in combination resulted in only modest increase in apoptosis. Evaluation of downstream signaling pathways indicated that Akt and STAT5 activity was not changed, but a delayed increase in MAPK activity was seen at high concentrations of SKI-606. SKI-606 inhibited normal progenitor proliferation to a lesser extent than imatinib. SKI-606 effectively inhibits Bcr-Abl and Src kinase activity and inhibits CML progenitor growth with relatively little effect on normal progenitors. However, SKI-606 does not demonstrate increased ability to eliminate primitive CML progenitors by apoptosis compared with imatinib, emphasizing the need for additional strategies besides Bcr-Abl kinase inhibition for curative therapy of CML.

In the present study we demonstrate that a murine proximal tubular cell line (MCT cells), expressing angiotensin II (ANG II) receptors [dissociation constant (Kd) = 0.89 nM; receptor density (R0) = 46,900 receptors/cell] in culture, can be induced to hypertrophy after the daily addition of exogenous ANG II (10(-8) M). This hypertrophic response was characterized by an increase in total cellular protein content, by an enhancement of [3H]leucine incorporation into precipitable proteins, and by an augmentation in cell size by cytofluorography. This ANG II effect producing MCT cell enlargement was demonstrable in the absence of cellular proliferation. Proliferation of MCT cells, however, could be induced by epidermal growth factor (EGF) or platelet-derived growth factor (PDGF), and pretreatment of rested MCT cells with ANG II further enhanced EGF-induced cell division. ANG II-induced hypertrophy in MCT cells was factor specific, in that it could be blocked with saralasin, and not induced by angiotensin I (ANG I). This hypertrophic response was also independent of prostaglandin E2 synthesis but was transducible by pertussis toxin-sensitive G proteins and involved, to some extent, the activation of Na(+)-H+ exchange. ANG II, as well as EGF and/or PDGF, moreover, could induce the cellular oncogenes c-fos, c-myc, c-N-ras, but not c-cis, which suggests that early gene activation is probably not a specific prerequisite for hypertrophy. Our findings demonstrate that ANG II, in culture, can be a single-factor event capable of inducing hypertrophy in proximal tubular cells.

Renal cell carcinoma is a highly malignant and often fatal disease of the kidney. It is difficult to treat, often because metastases are common at the time of presentation. Platelet-derived growth factor-D (PDGF-D) is a newly discovered member of the PDGF family; its function in tumor progression is largely unknown. Here, we examined the expression level of PDGF-D in human renal cell carcinoma by immunohistochemical staining using tissue arrays. We showed that human renal cell carcinoma expresses high levels of PDGF-D protein. The human renal cell carcinoma cell line SN12-C was stably transfected with pdgf-d cDNA. Overexpression of PDGF-D in SN12-C cells promoted tumor growth, angiogenesis, and metastasis of human renal cell carcinoma in an orthotopic severe combined immunodeficient (SCID) mouse model. PDGF-D overproduction in SN12-C cells increased the proliferation and migration of mural cells in vitro and improved perivascular cell coverage in vivo. Overexpression of PDGF-D led to increased expression of angiopoietin-1 and matrix metalloproteinase-9 in tumor tissues. ShRNAi and Gleevec were used to block PDGF-D expression and PDGF receptor beta (PDGFRbeta) signaling. Inhibition of PDGF-D expression by short hairpin RNA interference (shRNAi) and blockage of PDGFRbeta signaling by Gleevec inhibited the growth and lung metastasis of SN12-C cells grown orthotopically in SCID mice. Thus, PDGF-D is a potential candidate for controlling the progression of metastatic renal cell carcinoma. This opens up an avenue of investigation into novel therapeutic strategies for the treatment of renal cell carcinoma, including the use of recently developed tyrosine kinase inhibitors, such as Gleevec, which inhibit PDGF activity through inhibition of its receptor tyrosine kinase.

CP-673,451 is a potent inhibitor of platelet-derived growth factor beta-receptor (PDGFR-beta) kinase- and PDGF-BB-stimulated autophosphorylation of PDGFR-beta in cells (IC(50) = 1 nmol/L) being more than 450-fold selective for PDGFR-beta versus other angiogenic receptors (e.g., vascular endothelial growth factor receptor 2, TIE-2, and fibroblast growth factor receptor 2). Multiple models have been used to evaluate in vivo activity of CP-673,451 and to understand the pharmacology of PDGFR-beta inhibition and the effect on tumor growth. These models include an ex vivo measure of PDGFR-beta phosphorylation in glioblastoma tumors, a sponge model to measure inhibition of angiogenesis, and multiple models of tumor growth inhibition. Inhibition of PDGFR-beta phosphorylation in tumors correlates with plasma and tumor levels of CP-673,451. A dose of 33 mg/kg was adequate to provide >50% inhibition of receptor for 4 hours corresponding to an EC(50) of 120 ng/mL in plasma at C(max). In a sponge angiogenesis model, CP-673,451 inhibited 70% of PDGF-BB-stimulated angiogenesis at a dose of 3 mg/kg (q.d. x 5, p.o., corresponding to 5.5 ng/mL at C(max)). The compound did not inhibit vascular endothelial growth factor- or basic fibroblast growth factor-induced angiogenesis at concentrations which inhibited tumor growth. The antitumor efficacy of CP-673,451 was evaluated in a number of human tumor xenografts grown s.c. in athymic mice, including H460 human lung carcinoma, Colo205 and LS174T human colon carcinomas, and U87MG human glioblastoma multiforme. Once-daily p.o. x 10 days dosing routinely inhibited tumor growth (ED(50) < or = 33 mg/kg). These data show that CP-673,451 is a pharmacologically selective PDGFR inhibitor, inhibits tumor PDGFR-beta phosphorylation, selectively inhibits PDGF-BB-stimulated angiogenesis in vivo, and causes significant tumor growth inhibition in multiple human xenograft models.

Aberrant receptor tyrosine kinase signaling plays an important role in the molecular pathogenesis of brain tumors. We have been studying a previously identified human glioblastoma-derived PDGFR-alpha mutant that has an in-frame deletion in the extracellular domain, causing loss of exons 8 and 9 (PDGFR-alpha(delta8,9)). In the primary tumor, this deletion mutant receptor was shown to be amplified and overexpressed. The purpose of this study was to determine the expression, activity, localization, and transformation properties of this deletion mutant. In the absence of serum, or PDGF-AA, PDGFR-alpha(delta8,9) was phosphorylated on tyrosine residues, indicating ligand-independent autoactivation. Localization by staining and cell surface biotinylation studies revealed expression of the deletion mutant predominantly in the cytoplasm, with very little present on the cell surface. To determine if PDGFR-alpha(delta8,9) was oncogenic, we transfected wild-type and mutant receptors into Rat1 cells and performed analyses of cell growth, in vitro transformation, and subcutaneous growth in the nude mouse. PDGFR-alpha(delta8,9)-expressing cells displayed enhanced cell growth and survival in low serum, and formed foci in monolayer cultures. PDGFR-alpha(delta8,9)-expressing Rat1 cells were also tumorigenic when injected subcutaneously into nude mice. Expression of PDGFR-alpha(delta8,9) was also associated with increased c-Jun phosphorylation in the absence of PDGF ligand, demonstrating also that the mutant receptor is associated with altered intracellular signaling. These data demonstrate that PDGFR-alpha(delta8,9) is transforming, and it is the first demonstration of a naturally occurring tumor-derived mutant PDGFR-alpha with oncogenic properties.

Alveolar macrophages from normal individuals and patients with interstitial lung diseases spontaneously expressed a 4.2-kilobase mRNA complementary to the c-sis gene, a proto-oncogene coding for one of the chains of platelet-derived growth factor (PDGF). Concomitantly, these cells released a mediator with the properties of PDGF, including: chemotactic factor for smooth muscle cells whose activity was resistant to heat and acid, but sensitive to reduction; mitogenic (competence) activity for fibroblasts; ability to compete with PDGF for its receptor; and precipitated by an anti-PDGF antibody. While blood monocytes did not contain c-sis mRNA transcripts, monocytes matured in vitro expressed c-sis, consistent with the concept that expression of c-sis occurs during the differentiation of monocytes into alveolar macrophages. Together with the known actions of PDGF, these observations suggest that the c-sis proto-oncogene and its PDGF product are part of the armamentarium available to the alveolar macrophages for normal lung defense and participation in lung inflammation.

Bombesin and the related mammalian peptides, such as gastrin-releasing peptide (GRP), are potent mitogens for some fibroblast cell lines. Here we have examined the bombesin- and GRP-mediated changes in the phosphorylation of proteins in Swiss 3T3 cells and compared these to the events observed after platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and tumor promoter treatment. In agreement with previous reports, bombesin, GRP and PDGF, but not EGF, increased the activity of protein kinase C. This was assayed by an inhibition of [125I]EGF binding, stimulation in phosphorylation of pp60c-src on serine 12 and stimulation in phosphorylation of a group of 80 kd proteins. The different phosphorylated forms of the 80 kd proteins were examined by tryptic peptide mapping and shown to contain multiple phosphorylation sites. An investigation of the tyrosine phosphorylation events following mitogen treatment revealed a significant difference between PDGF and the bombesin peptides. PDGF treatment caused a marked increase in total cellular phosphotyrosine levels, and tyrosine phosphorylation both of known substrates and its own receptor. In contrast, bombesin and GRP treatments resulted in only a weak or undetectable increase in tyrosine phosphorylation of total cellular protein or known substrates. In this respect bombesin and GRP were more similar to EGF. The fact that the bombesin peptides do not induce a phosphorylation response identical with either PDGF or EGF suggests that there is not a single common signal pathway which is activated by all these mitogens.

The neuronal glutamate transporter, EAA<em>C</em>1, appears to both limit spillover between excitatory synapses and provide precursor for the synthesis of the inhibitory neurotransmitter, gamma-aminobutyric acid. There is evidence for a large intracellular pool of EAA<em>C</em>1 from which transporter is redistributed to the cell surface following activation of protein kinase <em>C</em> (PK<em>C</em>) or platelet-derived growth factor (<em>PDGF</em>) receptor by seemingly independent pathways. A variety of biotinylation strategies were employed to measure trafficking of EAA<em>C</em>1 to and from the plasma membrane and to examine the effects of phorbol ester and <em>PDGF</em> on these events. Biotinylation of cell surface protein under trafficking-permissive conditions (37 degrees <em>C</em>) resulted in a 2-fold increase in the amount of biotinylated EAA<em>C</em>1 within 15 min in <em>C</em>6 glioma and in primary neuronal cultures, suggesting that EAA<em>C</em>1 has a half-life of approximately 5-7 min for residence at the plasma membrane. Both phorbol ester and <em>PDGF</em> increased the amount of transporter labeled under these conditions. Using a reversible biotinylation strategy, a similarly rapid internalization of EAA<em>C</em>1 was observed in <em>C</em>6 glioma. Phorbol ester, but not <em>PDGF</em>, blocked this measure of internalization. Incubation at 18 degrees <em>C</em>, which blocks some forms of intracellular membrane trafficking, inhibited PK<em>C</em>- and <em>PDGF</em>-dependent redistribution of EAA<em>C</em>1 but had no effect on basal trafficking of EAA<em>C</em>1. These studies suggest that both PK<em>C</em> and <em>PDGF</em> accelerate delivery of EAA<em>C</em>1 to the cell surface and that PK<em>C</em> has an additional effect on endocytosis. The data also suggest that basal and regulated pools of EAA<em>C</em>1 exist in distinct compartments.

Platelet-derived growth factor (PDGF) is a critical regulator of proliferation and migration for mesenchymal type cells. In this study, we examined the role of mitogen-activated protein (MAP) kinases in the PDGF-BB-induced proliferation and migration of human adipose tissue-derived mesenchymal stem cells (hATSCs). The PDGF-induced proliferation was prevented by a pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor, SP600125. However, it was not prevented by a pretreatment with a p38 MAP kinase inhibitor, SB202190, and a specific inhibitor of the upstream kinase of extracellular signal-regulated kinase (ERK1/2), U0126. Treatment with PDGF induced the activation of JNK and ERK in hATSCs, and pretreatment with SP600125 specifically inhibited the PDGF-induced activation of JNK. Treatment with PDGF induced the cell cycle transition from the G0/G1 phase to the S phase, the elevated expression of cyclin D1, and the phosphorylation of Rb, which were prevented by a pretreatment with SP600125. In addition, the PDGF-induced migration of hATSCs was completely blocked by a pretreatment with SP600125, but not with U0126 and SB202190. These results suggest that JNK protein kinase plays a key role in the PDGF-induced proliferation and migration of mesenchymal stem cells.

Inhibition of multiple myeloma (MM) plasma cells in their permissive bone marrow microenvironment represents an attractive strategy for blocking the tumor/vessel growth associated with the disease progression. However, target specificity is an essential aim of this approach. Here, we identified platelet-derived growth factor (PDGF)-receptor beta (PDGFRbeta) and pp60c-Src as shared constitutively activated tyrosine-kinases (TKs) in plasma cells and endothelial cells (ECs) isolated from MM patients (MMECs). Our cellular and molecular dissection showed that the PDGF-BB/PDGFRbeta kinase axis promoted MM tumor growth and vessel sprouting by activating ERK1/2, AKT, and the transcription of MMEC-released proangiogenic factors, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). Interestingly, pp60c-Src TK-activity was selectively induced by VEGF in MM tumor and ECs, and the use of small-interfering (si)RNAs validated pp60c-Src as a key signaling effector of VEGF loop required for MMEC survival, migration, and angiogenesis. We also assessed the antitumor/vessel activity of dasatinib, a novel orally bioactive PDGFRbeta/Src TK-inhibitor that significantly delayed MM tumor growth and angiogenesis in vivo, showing a synergistic cytotoxicity with conventional and novel antimyeloma drugs (ie, melphalan, prednisone, bor-tezomib, and thalidomide). Overall data highlight the biologic and therapeutic relevance of the combined targeting of PDGFRbeta/c-Src TKs in MM, providing a framework for future clinical trials.

Activation of 44 and 42 kDa extracellular signal-regulated kinases (ERK)1/2 by angiotensin II (angII) plays an important role in vascular smooth muscle cell (VSMC) function. The dual specificity mitogen-actived protein (MAP) kinase/ERK kinase (MEK) activates ERK1/2 in response to angII, but the MEK activating kinases remain undefined. Raf is a candidate MEK kinase. However, a kinase other than Raf appears responsible for angII-mediated signal transduction because we showed previously that treatment with 1 microM phorbol 12, 13-dibutyrate (PDBU) for 24 h completely blocked Raf-Ras association in VSMC but did not inhibit activation of MEK and ERK1/2 by angII. We hypothesized that an atypical protein kinase C (PKC) isoform, which lacks a phorbol ester binding domain, mediated ERK1/2 activation by angII. Western blot analysis of rat aortic VSMC with PKC isoform-specific antibodies showed PKC-alpha, -beta1, -delta, -epsilon, and -zeta in relative abundance. All isoforms except PKC-zeta were down-regulated by 1 microM PDBU for 24 h suggesting that PKC-zeta was responsible for angII-mediated ERK1/2 activation. In response to angII, PKC-zeta associated with Ras as shown by co-precipitation of PKC-zeta with anti-H-Ras antibody. To characterize further the role of PKC-zeta, PKC-zeta protein was depleted specifically by transfection with antisense PKC-zeta oligonucleotides. Antisense PKC-zeta oligonucleotide treatment significantly decreased PKC-zeta protein expression (without effect on other PKC isoforms) and angII-mediated ERK1/2 activation in a concentration-dependent manner. In contrast, ERK1/2 activation by platelet-derived growth factor and phorbol ester was not significantly inhibited. These results demonstrate an important difference in signal transduction by angII compared with PDGF and phorbol ester in VSMC, and suggest a critical role for PKC-zeta and Ras in angII stimulation of ERK1/2.

A phosphatidylinositol-3 (PI-3) kinase activity of unknown biological function associates with tyrosine kinase-containing proteins, including a number of growth factor receptors after ligand stimulation. In the beta platelet-derived growth factor (beta PDGF) receptor, phosphorylation of a specific tyrosine residue within the kinase insert domain was required for its interaction with this enzyme. We show that substitutions of phenylalanine for tyrosine residue 731 or 742 within the kinase insert domain of the alpha PDGF receptor do not impair PDGF-induced tyrosine phosphorylation of the receptor or of an in vivo substrate, phospholipase C-gamma. Moreover, phosphatidylinositol turnover in response to ligand stimulation is unaffected. However, both lesions markedly impair receptor association with PI-3 kinase. Antiphosphotyrosine antibody-recoverable PI-3 kinase was also dramatically reduced in PDGF-stimulated cells expressing either mutant receptor. Since neither mutation abolished PDGF-induced mitogenesis or chemotaxis, we conclude that alpha PDGF receptor-associated PI-3 kinase activity is not required for either of these major PDGF signalling functions.

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.

Platelet-derived growth factor (PDGF) is a basic protein of relative molecular mass 30,000 (Mr 30K) composed of two polypeptide chains, designated PDGF A and PDGF B. The B-chain is encoded by the c-sis gene, the cellular counterpart of the simian sarcoma virus transforming gene v-sis. The PDGF A-chain cDNA clones recently isolated and sequenced from a transformed human clonal glioma cell line represent at least two alternatively spliced transcript species differing by 69 base pairs at the C-terminus. Here we demonstrate that the normal human umbilical vein endothelial cell (EC) A chain precursor lacks the 15 carboxy-terminal, highly basic amino acids encoded by the larger tumour cell cDNA. Surprisingly, culture media from monkey kidney cells (COS) transfected with the endothelial cDNA clone contained much less mitogenic activity than media from cells transfected with the longer tumour cell-derived A-chain cDNA. This functional difference appeared to be due to inefficient assembly or secretion of the recombinant endothelial-type growth factor. This suggests that some transformed cells may use alternative RNA splicing to modify normal growth factors and by so doing increase the efficiency of mitogen assembly or secretion.

The signaling activity of Platelet-derived growth factors A and B (PDGF-A and PDGF-B) that is mediated through the two receptor kinases, PDGFR-alpha and PDGFR-beta has been shown to be critical for the development of the cardiovascular organs, the kidney, the lung and the central nervous system. During the cloning of genes for VEGF related proteins, we isolated a mouse cDNA that can encode for a protein of 345 amino acids. A comparison of the amino acid sequence reveals that this predicted gene product displays 95% identity to human PDGF-C. The mouse Pdgfc gene maps to a region of chromosome 17 that is syntenic to human chromosome 6p21.3 In E9. 5-E15.5 mouse embryo, Pdgfc is widely expressed in the surface ectoderm and later in the germinal layer of the skin, the olfactory and otic placode and their derivatives and the lining of the oral cavity. In the gut and visceral organs, such as the lung and the kidney, Pdgfc mRNA is first expressed in the endodermal epithelium and later in mesenchymal tissues associated with the endodermal structures. Similar to other PDGFs, Pdgfc is widely expressed in mesenchymal precursors and the myoblast of the smooth and skeletal muscles. Contrary to PDGF-A, Pdgfc is not expressed in the central nervous system, except in the cerebellum, and neurogenic derivatives of the neural crest cells. Pdgfc is also absent from the heart and the vascular endothelium

OBJECTIVE

The role of growth factors and inflammation in regulating retinal pigment epithelial (RPE) function is complex and still poorly understood. The present study investigated human RPE cell proliferation and migration mediated by platelet-derived growth factor (PDGF) and inflammatory cytokines.

METHODS

Human fetal RPE (hfRPE) cells were obtained as previously described. Gene expressions of PDGF isoforms and their receptors were detected using real-time PCR. Protein expression, activity, and localization of PDGFR-alpha and -beta were analyzed by Western blot and immunohistochemistry. BrdU incorporation and wound healing assays were used to test the effects of different PDGF isoforms and inflammatory cytokines on hfRPE proliferation and migration. Annexin-V and phalloidin staining were used to detect apoptosis and the actin cytoskeleton, respectively.

RESULTS

PDGF-C and PDGF-D proteins are expressed in native human adult RPE, and mRNA levels are up to 100-fold higher than PDGF-A and -B. PDGFR-alpha and -beta proteins are expressed in native adult RPE and hfRPE (mainly localized to the apical membrane). In hfRPE, these receptors can be activated by PDGF-CC and -DD. PDGF-CC, -DD, and -BB significantly increased hfRPE proliferation, whereas PDGF-DD, -BB, and -AB significantly increased cell migration. An inflammatory cytokine mixture (TNF-alpha/IL-1beta/IFN-gamma) completely inhibited the stimulatory effect of PDGF-BB, -CC, and -DD; in contrast, this mixture stimulated the proliferation of choroidal cells. This inflammatory cytokine mixture also induced apoptosis, significant disruption of actin filaments and zonula occludens (ZO-1), and a decrease in transepithelial resistance.

CONCLUSIONS

These results suggest that proinflammatory cytokines in vivo can inhibit the proliferative effect of PDGF on human RPE and, at the same time, stimulate the proliferation of choroidal cells. They also suggest an important role of proinflammatory cytokines in overcoming local proliferative/wound-healing responses, thereby controlling the development of disease processes at the retina/RPE/choroid interface.

The low density lipoprotein receptor-related protein 1 (LRP1) has been implicated in intracellular signaling functions as well as in lipid metabolism. Recent in vivo and in vitro studies suggest that LRP1 is a physiological modulator of the platelet-derived growth factor (PDGF) signaling pathway. Here we show that in mouse fibroblasts LRP1 modulates PDGF-BB signaling by controlling endocytosis and ligand-induced down-regulation of the PDGF receptor beta (PDGFRbeta). In LRP1-deficient fibroblasts, basal PDGFRbeta tyrosine kinase activity was derepressed, and PDGF-BB-induced endocytosis and degradation of PDGFRbeta were accelerated as compared with control cells. This was accompanied by rapid uptake of receptor-bound PDGF-BB into the cells and by attenuated ERK activation in response to PDGF-BB stimulation. Pulse-chase analysis indicated that the steady-state turnover rate of PDGFRbeta was also accelerated in LRP-deficient fibroblasts. The rapid degradation of PDGFRbeta in the LRP1-deficient fibroblasts was prevented by MG132 and chloroquine. Furthermore, the association of PDGFRbeta with c-Cbl, a ubiquitin E3-ligase, as well as the ligand-induced ubiquitination of PDGFRbeta were increased in LRP1-deficient fibroblasts. We show that LRP1 can directly interact with c-Cbl, suggesting a Sprouty-like role for LRP1 in regulating the access of the PDGFRbeta to the ubiquitination machinery. Thus, LRP1 modulates PDGF signaling by controlling ubiquitination and endocytosis of the PDGFRbeta.

Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.

OBJECTIVE

Rapid induction of β-PDGF receptor (β-PDGFR) is a core feature of hepatic stellate cell activation, but its cellular impact in vivo is not well characterized. We explored the contribution of β-PDGFR-mediated pathway activation to hepatic stellate cell responses in liver injury, fibrogenesis, and carcinogenesis in vivo using genetic models with divergent β-PDGFR activity, and assessed its prognostic implications in human cirrhosis.

METHODS

The impact of either loss or constitutive activation of β-PDGFR in stellate cells on fibrosis was assessed following carbon tetrachloride (CCl4) or bile duct ligation. Hepatocarcinogenesis in fibrotic liver was tracked after a single dose of diethylnitrosamine (DEN) followed by repeated injections of CCl4. Genome-wide expression profiling was performed from isolated stellate cells that expressed or lacked β-PDGFR to determine deregulated pathways and evaluate their association with prognostic gene signatures in human cirrhosis.

RESULTS

Depletion of β-PDGFR in hepatic stellate cells decreased injury and fibrosis in vivo, while its auto-activation accelerated fibrosis. However, there was no difference in development of DEN-induced pre-neoplastic foci. Genomic profiling revealed ERK, AKT, and NF-κB pathways and a subset of a previously identified 186-gene prognostic signature in hepatitis C virus (HCV)-related cirrhosis as downstream of β-PDGFR in stellate cells. In the human cohort, the β-PDGFR signature was not associated with HCC development, but was significantly associated with a poorer outcome in HCV cirrhosis.

CONCLUSIONS

β-PDGFR is a key mediator of hepatic injury and fibrogenesis in vivo and contributes to the poor prognosis of human cirrhosis, but not by increasing HCC development.

Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3' untranslated region (3'-UTR) of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC) showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3' UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through activator protein 1 (AP-1), suggests a unique bimodal level of complexity previously unreported for disease-associated variants.

The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) and the metastatic phenotype are not very well defined. However, some of the genes involved in this process and their transcriptional regulation are beginning to be elucidated. For example, the switch from RGP to VGP and the metastatic phenotype is associated with loss of the AP-2α transcription factor. AP-2α regulates the expression of c-KIT, MMP-2, VEGF, and the adhesion molecule MCAM/MUC18. Recently, we reported that AP-2α also regulates two G-protein coupled receptors (GPCRs) PAR-1 and PAFR. In turn, the thrombin receptor, PAR-1, regulates the expression of the gap junction protein Connexin-43 and the tumor suppressor gene Maspin. Activation of PAR-1 also leads to overexpression and secretion of proangiogenic factors such as IL-8, uPA, VEGF, PDGF, as well certain integrins. PAR-1 also cooperates with PAFR to regulate the expression of the MCAM/MUC18 via phosphorylation of CREB. The ligands for these GPCRs, thrombin and PAF, are secreted by stromal cells, emphasizing the importance of the tumor microenvironment in melanoma metastasis. The metastatic phenotype of melanoma is also associated with overexpression and function of CREB/ATF-1. Loss of AP-2α and overexpression of CREB/ATF-1 results in the overexpression of MCAM/MUC18 which by itself contributes to melanoma metastasis by regulating the inhibitor of DNA binding-1 (Id-1). CREB/ATF-1 also regulates the angiogenic factor CYR-61. Our recent data indicate that CREB/ATF-1 regulates the expression of AP-2α, thus, supporting the notion that CREB is an important "master switch" in melanoma progression.

MicroRNAs are short noncoding, endogenous RNA species that posttranscriptionally inhibit gene expression by targeting the untranslated region (UTR) of mRNAs. Recently, it was shown that miR-29 inhibits expression of extracellular matrix proteins such as collagens, suggesting an antifibrotic function of miR-29. In the present study, we now investigated the role of miR-29 in profibrogenic growth factor expression as a further central mechanism of fibrosis. Screening of databases revealed putative miR-29 target sequences in the mRNA of platelet-derived growth factor (PDGF)-B, PDGF-B receptor, PDGF-C, vascular endothelial growth factor-A, and insulin-like growth factor (IGF)-I. To analyze miR-29 interaction with the predicted binding sites, we cloned the 3'-UTR sequences of the putative targets in fusion to the luciferase-reporter coding sequence. Functional miR-29 binding to PDGF-C and IGF-I mRNA sequences, but not to the corresponding mutants, was then proven by reporter assays. Hepatic stellate cells (HSC) that transdifferentiate into myofibroblasts, producing extracellular matrix proteins and profibrogenic growth factors, for example, the members of the PDGF family, are crucial for liver fibrosis. Myofibroblastic transition of primary HSC resulted in the loss of miR-29, but in a significant increase of PDGF-C and IGF-I. Compensation of reduced miR-29 levels by miR-29 overexpression in myofibroblastic HSC was followed by a definitive repression of IGF-I and PDGF-C synthesis. After experimental fibrosis, induced by bile-duct occlusion, miR-29 expression was shown to be reduced, but IGF-I and PDGF-C expression was upregulated, correlating inversely to the miR-29 pattern. Thus, we conclude that miR-29, downregulated during fibrosis, acts as an antifibrogenic mediator not only by targeting collagen biosynthesis, but also by interfering with profibrogenic cell communication via PDGF-C and IGF-I.

We have previously shown that after peripheral nerve lesion the synthesis of NGF is induced in cells of the nerve sheath (Heumann et al., 1987a). Further analysis led to the identification of growth factors and intracellular mechanisms responsible for this induction in sciatic fibroblasts (Lindholm et al., 1988; Hengerer et al., 1990). The present work aimed at the elucidation of the regulation of NGF synthesis in Schwann cells. A variety of cytokines and peptide growth factors, including interleukin-1 (IL-1) and platelet-derived growth factor (PDGF), which are known to increase NGF-mRNA in fibroblasts and astrocytes, failed to do so in Schwann cell cultures. Forskolin (FK), an activator of adenylate cyclase, increased the level of NGF-mRNA eightfold within 3 hr of incubation. The effect of FK on NGF-mRNA was mimicked by analogs of cAMP but not by dideoxyforskolin, an FK derivative not activating adenylate cyclase. Application of norepinephrine and isoproterenol also augmented the NGF-mRNA content. Pretreatment of Schwann cells with N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-8), an inhibitor of cyclic-nucleotide-dependent protein kinases, decreased both basal and elevated levels of NGF-mRNA. Ionomycin, a Ca2+ ionophore, and phorbol 12-myristate 13-acetate (TPA), an activator of protein kinase C, potentiated the effect of FK in an H-8-sensitive manner. We show that the action of FK is independent of changes in mRNA stability and of protein synthesis. Thus, in cultured Schwann cells upregulation of NGF-mRNA expression seems to be mainly achieved by a cAMP-triggered transcriptional activation of the NGF gene. Another striking difference between various glial cell types was revealed by application of transforming growth factor beta-1 (TGF-beta 1), which is the strongest inducer of NGF-mRNA in cultured astrocytes (Lindholm et al., 1990). Schwann cells responded to TGF-beta 1 by decreasing basal as well as FK-induced NGF-mRNA levels. Together with previously published work, our results show that cell-type-specific mechanisms not only account for the different control of NGF expression in neurons as compared to glial cells, but also reveal a surprising specificity of regulatory mechanisms in different non-neuronal cell types, even those derived from the same tissue such as fibroblasts and Schwann cells of peripheral nerves.