The role of small G proteins of the ADP-ribosylation factor (ARF) and Rho families on the activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF) and phorbol esters (PMA) has been investigated. The activation of PLD by PDGF and PMA was blocked by brefeldin A (BFA), an inhibitor of ARF activation, but not by Clostridium botulinum C3 exotoxin, an inhibitor of the activity of Rho. PDGF and PMA, in the presence of GTPgammaS, promoted the association of ARF and RhoA with cell membranes. Cells depleted of ARF and Rho by digitonin permeabilization showed a significant reduction of the activity of phospholipase D. Recombinant ARF was sufficient to restore agonist-induced PLD activity to digitonin-permeabilized, cytoplasm-depleted cells. In contrast, isoprenylated recombinant RhoA had no effects in this reconstitution assay. HIRcB cells were transiently transfected with wild-type and dominant-negative mutants of ARF1 and ARF6. Neither wt-ARF1 nor wt-ARF6 had any effects on agonist-dependent PLD activity. However, dominant-negative ARF1 and ARF6 mutants blocked the stimulation of PLD by PDGF but only partially inhibited the effects of PMA. These results demonstrate that ARF rather than Rho proteins mediate the activation of PLD by PDGF and phorbol esters in HIRcB fibroblasts.

BACKGROUND

More than 50% of patients undergoing lifelong suppressive antiviral treatment for HIV-1 infection develop minor HIV-1-associated neurocognitive disorders. Neurological complications during HIV-1 infection are the result of direct neuronal damage by proinflammatory products released from HIV-1-infected or -uninfected activated lymphocytes, monocytes, macrophages, microglia and astrocytes. The specific pro-inflammatory products and their roles in neurotoxicity are far from clear. We investigated proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) of HIV-demented (HIV-D) and HIV-nondemented (HIV-ND) patients and studied their affect on neuroglial toxicity.

RESULTS

Bioplex array showed elevated levels of signatory chemokines or cytokines (IL-6, IFN-γ, CXCL10, MCP-1 and PDGF) in the CSF of HIV-D patients (n = 7) but not in that of HIV-ND patients (n = 7). Among the signatory cytokines and chemokines, CXCL10 was distinctly upregulated in-vitro in HIV-1 (NLENG1)-activated human fetal astrocytes, HIV-1 (Ba-L)-infected macrophages, and HIV-1 (NLENG1)-infected lymphocytes. Virus-infected macrophages also had increased levels of TNF-α. Consistently, human fetal astrocytes treated with HIV-1 and TNF-α induced the signatory molecules. CXCL10 in combination with HIV-1 synergistically enhanced neuronal toxicity and showed chemotactic activity (~ 40 fold) for activated peripheral blood mononuclear cells (PBMC), suggesting the intersection of signaling events imparted by HIV-1 and CXCL10 after binding to their respective surface receptors, CXCR4 and CXCR3, on neurons. Blocking CXCR3 and its downstream MAP kinase (MAPK) signaling pathway suppressed combined CXCL10 and HIV-1-induced neurotoxicity. Bryostatin, a PKC modulator and suppressor of CXCR4, conferred neuroprotection against combined insult with HIV-1 and CXCL10. Bryostatin also suppressed HIV-1 and CXCL10-induced PBMC chemotaxis. Although, therapeutic targeting of chemokines in brain may have adverse consequences on the host, current findings and earlier evidence suggest that CXCL10 could strongly impede neuroinflammation.

CONCLUSIONS

We have demonstrated induction of CXCL10 and other chemokines/cytokines during HIV-1 infection in the brain, as well as synergism of CXCL10 with HIV-1 in neuronal toxicity, which was dampened by bryostatin.

OBJECTIVE

We investigated the regulation of p38 mitogen-activated protein kinase (MAPK) by platelet-derived growth factor (PDGF)-BB and its biological effects in cultured normal and diabetic rat vascular smooth muscle cells (VSMCs).

RESULTS

VSMC growth from diabetic rats was faster than that from normal rats. The expression of the PDGF beta-receptor in diabetic VSMCs was significantly elevated compared with that in normal cells, and PDGF-BB-induced p38 phosphorylation in diabetic cells was more enhanced via MAPK kinase (MKK) 3/6. The level of PKC activity in diabetic cells increased more than that in normal cells with or without PDGF-BB. Although protein kinase C (PKC)-betaII and PKC-delta were activated by diabetes, PDGF-BB could further enhance the level of PKC-delta alone. PDGF-BB-induced cell migration was more elevated in diabetic VSMCs, and the increase was significantly inhibited by SB-203580, rottlerin, and antisense oligodeoxynucleotides for PKC-delta. PDGF-BB-induced p38 phosphorylation also regulated cell growth, cyclooxygenase-2 levels, and arachidonic acid release, but not apoptosis. These levels were more elevated in diabetic cells, which were inhibited by SB-203580.

CONCLUSIONS

Our study established that PDGF-BB phosphorylated p38 via PKC-delta and the subsequent MKK 3/6, leading to cell growth regulation and the progression of a chronic inflammatory process in diabetic VSMCs.

OBJECTIVE

Elevated apolipoprotein D (apoD) levels are associated with reduced proliferation of cancer cells. We therefore investigated whether apoD, which occurs free or associated with HDL, suppresses vascular smooth muscle cell (VSMC) proliferation, which is related to the pathobiology of disease.

RESULTS

Intense immunoreactivity for apoD was observed in human atherosclerotic plaque but not in normal coronary artery. However, an increase in apoD mRNA was seen in quiescent relative to proliferating fetal lamb aortic VSMCs, and in the rat aortic VSMC line (A10), we demonstrated uptake of apoD from serum. Stable transfection of apoD in A10 cells in the absence of serum did not influence VSMC proliferation assessed by [3H]-thymidine incorporation. ApoD, administered at a dose of 100 ng/mL, completely inhibited basal as well as platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation (P<0.01) but had no effect on fibroblast growth factor-induced VSMC proliferation. ApoD did not suppress PDGF-BB or fibroblast growth factor-2-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 but selectively inhibited PDGF-BB-mediated ERK1/2 nuclear translocation.

CONCLUSIONS

Our data suggest that apoD selectively modulates the proliferative response of VSMC to growth factors by a mechanism related to nuclear translocation of ERK1/2.

C<em>D</em>20 is a transmembrane protein that functions as a Ca(2+)-permeable cation channel (Bubien, J. K., Zhou, L. J., Bell, P. <em>D</em>., Frizzel, R. A., and Tedder, T. F. (1993) J. Cell Biol. 121, 1121-1132) and is involved in growth regulation of B lymphocytes. In order to further investigate the role of calcium entry in cell cycle progression, we introduced the c<em>D</em>NA encoding a Ca(2+)-permeable cation channel, C<em>D</em>20, into Balb/c 3T3 cells. Balb/c 3T3 cells transfected with a vector containing c<em>D</em>NA encoding C<em>D</em>20 expressed the C<em>D</em>20 protein, which was detected by assaying the binding of a monoclonal antibody against C<em>D</em>20. Calcium-permeable cation channel activity was detected in C<em>D</em>20-expressing cells by whole cell patch clamp recording and microfluorometric determination of the cytoplasmic Ca2+ concentration using fura-2. The expression of C<em>D</em>20 induced significant alterations in the responses of the cells to insulin-like growth factor-I (IGF-I). IGF-I induced <em>D</em>NA synthesis by control cells only when they had been pretreated with both platelet-derived growth factor (<em>PDGF</em>) and epidermal growth factor (EGF). In contrast, <em>D</em>NA synthesis by 30% of the quiescent C<em>D</em>20-expressing cells was initiated in response to IGF-I in the absence of priming with <em>PDGF</em> and EGF. When control quiescent cells were primed with <em>PDGF</em> and EGF, the addition of IGF-I led to the initiation of <em>D</em>NA synthesis after 14 h or more, whereas it induced <em>D</em>NA synthesis by C<em>D</em>20-expressing cells primed with <em>PDGF</em> and EGF 4 h earlier. The IGF-induced <em>D</em>NA synthesis was dependent on extracellular Ca2+, and expression of C<em>D</em>20 reduced the concentration of extracellular Ca2+ required for it. Furthermore, <em>D</em>NA synthesis by approximately 25% of the C<em>D</em>20-expressing cells was initiated after priming with <em>PDGF</em> and EGF, even in the absence of the progression factor IGF-I. These results indicate that C<em>D</em>20 expressed in Balb/c 3T3 cells functions as a constitutively active Ca(2+)-permeable cation channel and that expression of C<em>D</em>20 accelerates G1 progression in a Ca(2+)-dependent manner.

Evolving anti-vascular endothelial growth factor (VEGF) treatments for neovascular age-related macular degeneration (nAMD) include long acting agents, combination strategies involving new pathways, topical agents, sustained-release, and genetic therapy strategies. Areas covered: Brolucizumab and abicipar pegol have smaller molecular size, facilitating higher concentrations and potentially longer duration than current anti-VEGF agents. Agents being combined with anti-VEGFs include OPT-302 (to inhibit VEGF-C and VEGF-D); pegpleranib and rinucumab (to inhibit platelet derived growth factor, PDGF - but both failed to show consistently improved visual outcomes compared to anti-VEGF monotherapy); and RG7716, ARP-1536 and nesvacumab (to activate the Tie-2 tyrosine kinase receptor, which reduces permeability). X-82 is an oral anti-VEGF and anti-PDGF being tested in phase 2 studies. Topical anti-VEGF ± anti-PDGF drugs under study include pazopanib, PAN-90806, squalamine lactate, regorafinib, and LHA510. Sustained-release anti-VEGF delivery treatments, such as the ranibizumab Port Delivery System, GB-102, NT-503, hydrogel depot, Durasert, and ENV1305 aim to reduce the burden of frequent injections. Gene therapies with new viral vectors hold the potential to induce sustained expression of anti-angiogenic proteins via the retina's cellular apparatus, and include AVA-101/201, ADVM-202/302, AAV2-sFLT01, RGX314, and Retinostat. Expert opinion: There are many emerging anti-VEGF treatments that aim to improve visual outcomes and reduce the treatment burden of nAMD.

Injuries to the avascular region of the meniscus occur frequently and may be difficult to repair. This study was designed to determine whether growth factors could diffuse from a collagen sponge or a collagen gel into meniscal tissue and stimulate healing of defects using an in vitro model. The diffusion of platelet-derived growth factor (PDGF) from the collagen carriers into the medium was rapid with approximately 50% being released from the collagen sponge within the first hour. After 5 d of incubation, 8% of the PDGF was present in the meniscus, 11% in the collagen sponge, and 62% had been released into the medium. Similar results were obtained when a collagen gel was used as a carrier. Histological evaluation of the meniscal explants after 2 wk in culture revealed extensive proteoglycan staining in the areas surrounding defects treated with either hepatocyte growth factor (HGF) or PDGF compared with controls without growth factor. The HGF-PDGF treatment resulted in alignment and migration of meniscal cells toward the defect, which was not observed in untreated controls. At 3-7 d, increased number of cells were observed in defects treated with collagen gels (but not the sponge) with PDGF-HGF. At 4 wk, combined HGF-PDGF treatment resulted in the formation of tissue with birefringence by polarized microscopy, suggestive of organized collagen. The data suggest that use of specific PDGF-HGF may enhance the repair of meniscal injuries.

Platelet-derived growth factors (PDGFs) play an integral role in normal tissue growth and maintenance as well as many human pathological states including atherosclerosis, fibrosis, and tumorigenesis. The PDGF family of ligands is comprised of A, B, C, and D chains. Here, we provide the first functional characterization of the PDGF-C promoter. We examined 797 bp of the human PDGF-C promoter and identified several putative recognition elements for Sp1, Ets Egr-1, and Smad. The proximal region of the PDGF-C promoter bears a remarkable resemblance to a comparable region of the PDGF-A promoter (1). Binding and transient transfection analysis in primary vascular smooth muscle cells revealed that PDGF-C, like PDGF-A, is under the transcriptional control of the zinc finger nuclear protein Egr-1 (early growth response-1). Electrophoretic mobility shift analysis using both smooth muscle cell nuclear extracts and recombinant protein revealed that Egr-1 and Sp1 bind this region of the PDGF-C promoter (Oligo C, -35 to -1). Egr-1 competes with Sp1 for overlapping binding sites even when the former is at a stoichiometric disadvantage. Reverse transcriptase PCR and supershift analysis demonstrate that fibroblast growth factor-2 (FGF-2) stimulates both Egr-1 and PDGF-C mRNA expression in a time-dependent and transient manner and that FGF-2-inducible Egr-1 binds the proximal PDGF-C promoter. FGF-2-inducible PDGF-C expression was completely abrogated using catalytic DNA (DNAzymes) targeting Egr-1 but not by its scrambled counterpart. Moreover, using pharmacological inhibitors we demonstrate the critical role of ERK but not JNK in FGF-2-inducible PDGF-C expression. These findings thus demonstrate that PDGF-C transcription, activated by FGF-2, is mediated by Egr-1 and its upstream kinase ERK.

Protein kinase <em>D</em> (PK<em>D</em>) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PK<em>D</em> isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PK<em>D</em>1 and PK<em>D</em>2 in various cellular contexts. This study shows that PK<em>D</em>1 and PK<em>D</em>2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The α(1)-adrenergic receptor agonist norepinephrine selectively activates PK<em>D</em>1, thrombin and <em>PDGF</em> selectively activate PK<em>D</em>2, and endothelin-1 and PMA activate both PK<em>D</em>1 and PK<em>D</em>2. PKC activity is implicated in the α(1)-adrenergic receptor pathway that activates PK<em>D</em>1 and the thrombin- and <em>PDGF</em>-dependent pathways that activate PK<em>D</em>2. Endothelin-1 activates PK<em>D</em> via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PK<em>D</em> activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PK<em>D</em> substrates in cardiomyocytes. We show that overexpression of an activated PK<em>D</em>1-S744E/S748E transgene increases CREB-Ser(133) and cTnI-Ser(23)/Ser(24) phosphorylation, but agonist-dependent pathways that activate native PK<em>D</em>1 or PK<em>D</em>2 selectively increase CREB-Ser(133) phosphorylation; there is no associated increase in cTnI-Ser(23)/Ser(24) phosphorylation. Gene silencing studies provide unanticipated evidence that PK<em>D</em>1 down-regulation leads to a compensatory increase in PK<em>D</em>2 activity and that down-regulation of PK<em>D</em>1 (alone or in combination with PK<em>D</em>2) leads to an increase in CREB-Ser(133) phosphorylation. Collectively, these studies identify distinct roles for native PK<em>D</em>1 and PK<em>D</em>2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure.

Platelet-derived growth factor (PDGF) is a polypeptide growth factor which has been implicated as a major mitogen involved in wound healing. The PDGF appears to promote periodontal regeneration; however, its distribution in gingival tissues is not known and how it participates in gingival wound healing is unclear. Using highly specific antibodies we have studied the distribution of PDGF A and B chains and alpha- and beta-PDGF receptors in healing human gingival wounds. Wounds were created by making a 0.75 mm deep incision in the papilla and healthy gingiva and biopsies were obtained from the same site after 8 h and 1, 3, 7, 14 and 21 d. Frozen sections were immunostained with affinity purified antibodies. The results showed that both epithelium and fibrin clot manifested positive immunostaining for anti-PDGF-A and B-chain antibodies. Staining was present in unwounded and wounded epithelia, and in the fibrin clot it appeared to be more intense for the PDGF-A chain. Blood vessels in connective tissue were also positive while other areas were largely negative. No significant staining was detectable in healthy tissues for anti-PDGF-alpha or -beta receptor antibodies. However, the wound site began to manifest positive immunostaining fro anti-beta-receptor antibody after 3 d of healing, became maximal at 7 d, and then decreased. Our data indicate, but do not prove, that gingival epithelium may be a source of PDGF A and B chains and that the A chain may have a more prominent role to play during early stages of healing. Expression of PDGF beta-receptor appears later at the wound site, indicating that the PDGF B isomer may regulate later wound healing events.

1 Sphingosine-1-phosphate (S1P) is considered a potent mitogen for mesangial cells and activates the classical mitogen-activated protein kinase (MAPK) cascade via S1P receptors. In this study, we show that S1P signalling is rapidly desensitized upon S1P receptor activation. A complete loss of S1P sensitivity occurs after 10 min of S1P pretreatment and remains for at least 8 h. A similar desensitization is also seen with the S1P mimetic FTY720-phosphate, but not with the nonphosphorylated FTY720, nor with sphingosine or ceramide. 2 Prestimulating the cells with extracellular ATP or UTP, which bind to and activate P2Y receptors on mesangial cells, a similar rapid desensitization of the S1P receptor occurs, suggesting a heterologous desensitization of S1P receptors by P2Y receptor activation. Furthermore, adenosine binding to P1 receptors triggers a similar desensitization. In contrast, two other growth factors, PDGF-BB and TGFbeta2, have no significant effect on S1P-induced MAPK activation. 3 S1P also triggers increased inositol trisphosphate (IP3) formation, which is completely abolished by S1P pretreatment but only partially by ATP pretreatment, suggesting that IP3 formation and MAPK activation stimulated by S1P involve different receptor subtypes. 4 Increasing intracellular cAMP levels by forskolin pretreatment has a similar effect on desensitization as adenosine. Moreover, a selective A3 adenosine receptor agonist, which couples to phospholipase C and increases IP3 formation, exerted a similar effect. 5 Pretreatment of cells with various protein kinase C (PKC) inhibitors prior to ATP prestimulation and subsequent S1P stimulation leads to a differential reversal of the ATP effect. Whereas the broad-spectrum protein kinase inhibitor staurosporine potently reverses the effect, the PKC-alpha inhibitor CGP41251, the PKC-delta inhibitor rottlerin and calphostin C show only a partial reversal at maximal concentrations. 6 Suramin, which is reported as a selective S1P3 receptor antagonist compared to the other S1P receptor subtypes, has no effect on the S1P-induced MAPK activation, thus excluding the involvement of S1P3 in this response. 7 In summary, these data document a rapid homologous and also heterologous desensitization of S1P signalling in mesangial cells, which is mechanistically triggered by PKC activation and eventually another staurosporine-sensitive protein kinase, as well as by increased cAMP formation.

Using a pharmacophore model for ATP-competitive inhibitors interacting with the active site of the EGF-R protein tyrosine kinase (PTK), 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines have been identified as a novel class of potent EGF-R protein tyrosine kinase inhibitors. In an interactive process, this class of compounds was then optimized. 13, 14, 28, 36, 37, and 44, the most potent compounds of this series, inhibited the EGF-R PTK with IC50 values in the low nanomolar range. High selectivity toward a panel of nonreceptor tyrosine kinases (c-Src, v-Abl) and serine/threonine kinases (PKC alpha, PKA) was observed. Kinetic analysis revealed competitive type kinetics relative to ATP. In cells, EGF-stimulated cellular tyrosine phosphorylation was inhibited by compounds 13, 36, 37, and 44 at IC50 values between 0.1 and 0.4 microM, whereas PDGF-induced tyrosine phosphorylation was not affected by concentrations up to 10 microM. In addition, these compounds were able to selectively inhibit c-fos mRNA expression in EGF-dependent cell lines with IC50 values between 0.1 and 2 microM, but did not affect c-fos mRNA induction in response to PDGF or PMA (IC50 >100 microM). Proliferation of the EGF-dependent MK cell line was inhibited with similar IC50 values. From SAR studies, a binding mode for 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines as well as for the structurally related 4-(phenylamino)quinazolines at the ATP-binding site of the EGF-R tyrosine kinase is proposed. 4-(Phenylamino)7H-pyrrolo[2,3-d]pyrimidines therefore represent a new class of highly potent tyrosine kinase inhibitors which preferentially inhibit the EGF-mediated signal transduction pathway and have the potential for further evaluation as anticancer agents.

OBJECTIVE

To explore further the significance of three patterns of cytokine dialogues that have been characterized between human corneal and limbal epithelial cells and fibroblasts.

METHODS

Northern hybridization of the transcript expression of type I cytokine receptors (EGFR, IL-1R, and PDGFR-beta), type II cytokines (bFGF, LIF, and TGF-beta 1), and type III cytokines (HGF and KGF) by human corneal and limbal fibroblasts was conducted under the modulation of TGF-alpha, PDGF-BB, IL-1 beta, and EGF (type I cytokines). The mechanism of upregulation by IL-1 beta was studied further with respect to proto-oncogene expression and under the treatment of cycloheximide and actinomycin D.

RESULTS

Results showed that EGF upregulated LIF and HGF but downregulated KGF and M-CSF. Unlike EGF, TGF-alpha upregulated additional EGFR, PDGFR-beta, bFGF, and TGF-beta 1, suggesting that although they share the same EGFR, TGF-alpha, which is produced by epithelium, is more effective in activating fibroblasts than EGF, which is present in tears. The upregulation of PDGF-BB was similar to that of TGF-alpha, except that it further stimulated IL-8, supporting their synergistic roles in promoting wound healing. Uniquely, IL-1 beta upregulated KGF expression by limbal fibroblasts more than corneal fibroblasts and IL-8 and M-CSF expression, but it downregulated PDGFR-beta. In IL-1 beta, the upregulation of cytokines and receptors was preceded by the upregulation of c-fos, c-jun, and c-myc, and it was inhibited by actinomycin D. Its upregulation of LIF was superinduced, but the upregulation of bFGF and KGF was inhibited, and that of the rest was not affected by cycloheximide.

CONCLUSIONS

These findings suggest that epithelial cells under stress or injury (producing IL-1) might preferentially activate limbal epithelial stem cells indirectly by fibroblasts and simultaneously might promote inflammation during wound healing.

The mitogenic action of cytokines such as epidermal growth factor (EGF) or platelet derived growth factor (PDGF) involves the stimulation of a signal cascade controlled by a small G protein called Ras. Mutations of Ras can cause its constitutive activation and, as a consequence, bypass the regulation of cell growth by cytokines. Both growth factor-induced and oncogenic activation of Ras involve the conversion of Ras from the GDP-bound (D-Ras) to the GTP-bound (T-Ras) forms. T-Ras activates a network of protein kinases including c-Mos, c-Raf-1 and MAP kinase. Eventually the activation of MAP kinase leads to the activation of the elongation factor 4E and several transcription factors such as c-Jun, c-Myc and c-Fos. There are several modulators of Ras activity, such as the GTPase activating proteins (GAP1 and NF1), which stimulate the conversion of T-Ras to D-Ras. A series of small NF1 fragments, which bind T-Ras, as well as truncated forms of derivatives of c-Raf-1, c-Jun and c-Myc, are capable of blocking the T-Ras-activated mitogenesis in a competitive manner. These agents offer a unique opportunity to control the proliferation of T-Ras-associated tumors, which represent more than 30% of total human carcinomas.

Previous studies have identified several cytokines as inducers of tenascin-C (TN-C) expression in various tissue culture systems. However, the signaling pathways of the regulation of TN-C expression are almost unknown. In this study, we clarified the molecular mechanism(s) underlying the regulation of the TN-C gene by platelet derived growth factor (PDGF) in cultured human dermal fibroblasts. PDGF induced the expression of TN-C protein as well as mRNA in a dose-dependent manner. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the PDGF-mediated upregulation of TN-C mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not. The PDGF-mediated induction of TN-C expression was inhibited by the treatment of fibroblasts with a selective phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, or LY294002. These results suggest that PDGF induced the expression of TN-C at a transcriptional level via phosphoinositide3-kinase/Akt signaling pathways. We performed serial 5' deletions and a transient transfection analysis to define the region in the TN-C promoter mediating the responsiveness to PDGF. Overexpression of Sp1, Ets1, or Ets2 activated the TN-C promoter and superinduced TN-C promoter activity stimulated by PDGF, whereas overexpression of Fli1 inhibited the effects of PDGF on TN-C expression. Mutation of the Sp1/3 binding sites or Ets binding sites in the TN-C promoter region responsible to PDGF abrogated the PDGF-inducible promoter activity. Immunoprecipitation analysis revealed that Sp1, Ets1, and Ets2 form a transcriptionally active complex. On the other hand, the interaction of Fli1 with Sp1 decreased after PDGF treatment. These results suggest that the upregulation of TN-C expression by PDGF involves Ets family transcription factors, co-operating with Sp1.

Cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats express both alpha and beta isoforms of the platelet-derived growth factor (PDGF) receptors at high levels (100,000 and 240,000 sites/cell, respectively). In this cell type, PDGF-BB elicited a mitogenic response; however, PDGF-AA increased only protein synthesis without activating DNA synthesis. Protein kinase C (PKC) was activated by PDGF-AA as well as PDGF-BB with concomitant translocation from cytosol to membrane fractions. However, the hypertrophic effect of PDGF-AA was not affected by depletion of cellular PKC, whereas the mitogenic action of PDGF-BB was partially attenuated by the depletion. Following incubation with PDGF-AA or -BB, phospholipase C-gamma 1 (PLC-gamma 1) and phosphatidylinositol 3-kinase were tyrosine phosphorylated; however, the phosphorylation of Ras-GTPase-activating protein was induced only by PDGF-BB. Both PDGF isoforms resulted in a prompt and transient increase in the level of 1,2-diacylglycerol (DAG), presumably through the action of PLC-gamma 1. After returning to basal levels, the rate of DAG synthesis steadily increased for at least 15 min due to activation of phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC). Incubation with PDGF-BB-activated phospholipase D (PLD) in a PKC-dependent manner resulting in the formation of phosphatidic acid (PA). PA was also formed by the sequential reactions of PC-PLC and DAG kinase in the PDGF-BB-stimulated VSMC, and these sequential reactions were not affected by PKC depletion. In contrast, PDGF-AA stimulation did not result in increased PA synthesis as neither PLD nor DAG kinase activities were affected. PA may be a significant second messenger in the activation of DNA synthesis by PDGF-BB. These results indicate that signaling mechanisms of the PDGF-alpha and -beta receptors in VSMC are distinctly different in signal transduction in VSMC and that the alpha receptor promotes cellular hypertrophy (but not hyperplasia), whereas a mitogenic response is mediated only through the beta receptor.

We have shown in bovine tracheal myocytes that extracellular signal-regulated kinase (ERK) and Rac1 function as upstream activators of transcription from the cyclin D(1) promoter. We now examine the role of phosphatidylinositol (PI) 3-kinase in this process. PI 3-kinase activity was increased by platelet-derived growth factor (PDGF) and attenuated by the PI 3-kinase inhibitors wortmannin and LY294002. These inhibitors also decreased cyclin D(1) promoter activity, protein abundance, and DNA synthesis. Overexpression of the active catalytic subunit of PI 3-kinase (p110(PI) (3-K)CAAX) was sufficient to activate the cyclin D(1) promoter. Wortmannin and LY294002 failed to attenuate PDGF-induced ERK activation, and overexpression of p110(PI) (3-K)CAAX was insufficient to activate ERK. p110(PI) (3-K)CAAX-induced cyclin D(1) promoter activity was not blocked by PDD guanidine triphosphatase Rac1 regulate cyclin D(1) promoter activity by similar mechanisms. p110(PI) (3-K)CAAX-induced cyclin D(1) promoter activity was decreased by two inhibitors of Rac1-mediated signaling, catalase and diphenylene iodonium. Further, PDGF, PI 3-kinase, and Rac1 each activated the cyclin D(1) promoter at the cyclic adenosine monophosphate response element binding protein (CREB)/activating transcription factor (ATF)-2 binding site, as evidenced by expression of a CREB/ATF-2 reporter plasmid. Finally, PI 3-kinase and Rac1-induced CREB/ATF-2 transactivation were each inhibited by catalase. Together, these data suggest that in airway smooth muscle (ASM) cells, PI 3-kinase regulates transcription from the cyclin D(1) promoter and DNA synthesis in an ERK-independent manner. Further, PI 3-kinase and Rac1 regulate ASM cell cycle traversal via a common cis-regulatory element in the cyclin D(1) promoter.

Despite extensive analysis of phosphoinositide 3-hydroxykinases (PI 3-kinases) at the molecular level, comparatively little is known about the mechanisms by which products of these enzymes exert their expected second-messenger functions. This study examines the metabolism of D-3 phosphoinositides in mouse Ph-N2 fibroblasts lacking the platelet-derived growth factor (PDGF) alpha-receptor. Treatment of these cultures with BB PDGF, but not AA PDGF, resulted in transient activation of PI 3-kinase activity measured in vitro. Treatment of myo-[3H]inositol-labelled Ph-N2 cells with BB PDGF resulted in the rapid induction of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 and, to a smaller extent, PtdIns3P. The appearance of PtdIns(3,4,5)P3 preceded that of PtdIns(3,4)P2 and PtdIns3P after the addition of PDGF, suggesting that PtdIns(4,5)P2 is the preferred substrate of the agoniststimulated PI 3-kinase in intact cells. Treatment of both resting and PDGF-stimulated cells with the fungal metabolite wortmannin resulted in pronounced, selective effects on the levels of all D-3 phosphoinositides. Kinetic studies with this PI 3-kinase inhibitor revealed the presence of at least two independent routes for the biosynthesis of D-3 phosphoinositides in PDGF-treated cells.

BACKGROUND

Periodontal regeneration requires a coordinated series of events that includes not only the recruitment of periodontal ligament (PDL)-specific cells, but vascular cells as well. The mechanisms of action of enamel matrix derivative (EMD) are poorly understood, and its effects on vascular cells are unknown. The objective of this study was to examine the extent to which EMD affects angiogenesis and PDL cell recruitment.

METHODS

The effects of EMD on human microvascular endothelial cells (HMVECs) were determined by examining proliferation, chemotaxis, angiogenesis, and migration. Proliferation was determined using water-soluble tetrazolium salt (WST)-1 reagent. Chemotaxis was determined using microporous-culture well inserts. Angiogenesis was assessed on plates containing matrigel. The effects of HMVECs on the migration of PDL cells were assessed by evaluating PDL cell outgrowth from collagen gels cultured in the presence of HMVECs on fibrin matrix and surrounded by fibronectin-containing fibrin clots at 24 hours. Effects of EMD on PDL expression of vascular endothelial cell (VEGF) types (A, B, C, and D) and isoforms were determined using reverse transcription-polymerase chain reaction (RT-PCR). Production of VEGF, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and transforming growth factor (TGF)-beta1 by EMD-stimulated PDL cells was assessed quantitatively in conditioned media using specific enzyme-linked immunosorbent assays (ELISAs).

RESULTS

EMD at concentrations <50 microg/ml resulted in significant (P <0.05) stimulation of HMVEC proliferation. Compared to baseline, EMD also stimulated a 100% increase in HMVEC chemotaxis when PDL cells were present (P <0.05). All doses of EMD tested (25, 50, and 100 microg/ml) increased angiogenesis in vitro. HMVECs, in combination with EMD at a concentration of 100 microg/ml, stimulated a 750% increase in migration of PDL cells from collagen gels into fibrin clots compared to controls when neither was present. RT-PCR results indicated that PDL cells expressed VEGF-A, -B, and -C and multiple isoforms of VEGF-A, including VEGF(121), VEGF(165), and VEGF(189), whether or not EMD was present in the culture media. ELISAs determined a 400% increase in VEGF concentration by PDL C cells in EMD-stimulated conditioned media and a similar increase in TGF-beta(1)-stimulated media.

CONCLUSIONS

It is likely that EMD stimulates angiogenesis directly by stimulating endothelial cells and indirectly by stimulating the production of angiogenic factors (VEGF) by PDL cells. Importantly, the data are consistent with the concept that EMD enhances bidirectional communication between HMVEC and PDL cells during angiogenesis associated with healing.

Integrin recycling is critical for cell migration. Protein kinase D (PKD) mediates signals from the platelet-derived growth factor receptor (PDGF-R) to control αvβ3 integrin recycling. We now show that Rabaptin-5, a Rab5 effector in endosomal membrane fusion, is a PKD substrate. PKD phosphorylates Rabaptin-5 at Ser407, and this is both necessary and sufficient for PDGF-dependent short-loop recycling of αvβ3, which in turn inhibits α5β1 integrin recycling. Rab4, but not Rab5, interacts with phosphorylated Rabaptin-5 toward the front of migrating cells to promote delivery of αvβ3 to the leading edge, thereby driving persistent cell motility and invasion that is dependent on this integrin. Consistently, disruption of Rabaptin-5 Ser407 phosphorylation reduces persistent cell migration in 2D and αvβ3-dependent invasion. Conversely, invasive migration that is dependent on α5β1 integrin is promoted by disrupting Rabaptin phosphorylation. These findings demonstrate that the PKD pathway couples receptor tyrosine kinase signaling to an integrin switch via Rabaptin-5 phosphorylation.

PDGF isoforms are a family of polypeptides that bind to cell surface receptors and induce fibroblast proliferation and chemotaxis. The PDGF-A and -B chain isoforms have been implicated in fibroproliferative lung injury in animal models and in human disease. Two recently recognized PDGF polypeptides, PDGF-C and -D, differ from the PDGF-A and -B isoforms in that they require proteolytic cleavage before they can bind and activate the PDGF receptors. Our findings demonstrate that administration of bleomycin to murine lungs leads to a significant increase in PDGF-C mRNA expression and a significant decrease in PDGF-D mRNA expression. PDGF-C expression was localized to areas of lung injury by in situ hybridization, and PDGF-C expression was not upregulated in the lungs of BALB/c mice that are resistant to bleomycin-induced lung fibrosis. Moreover, there is in vivo phosphorylation of the PDGF-receptor that binds PDGF-C in response to bleomycin administration. These observations strongly suggest a role for PDGF-C in bleomycin-induced pulmonary fibrosis.

PD 166285, a novel protein tyrosine kinase inhibitor of a new structural class, the 6-aryl-pyrido[2,3-d]pyrimidines, was synthesized as the most potent and soluble analog of a series of small molecules originally identified by screening a compound library with assays that measured protein tyrosine kinase activity. PD 166285 was found to inhibit Src nonreceptor tyrosine kinase, fibroblast growth factor receptor-1, epidermal growth factor receptor and platelet-derived growth factor receptor beta subunit (PDGFR-beta), tyrosine kinases with half-maximal inhibitory potencies (IC50 values) of 8.4 +/- 2.3 nM (n = 6), 39.3 +/- 2.8 nM (n = 16), 87.5 +/- 13.7 nM (n = 6) and 98.3 +/- 7.9 nM (n = 16), respectively. PD 166285 also demonstrated inhibitory activity against mitogen-activated protein kinase (IC50 = 5 microM) and protein kinase C (IC50 = 22.7 microM). PD 166285 was further characterized as an ATP competitive inhibitor of Src nonreceptor tyrosine kinase, PDGFR-beta, fibroblast growth factor receptor-1 and epidermal growth factor receptor tyrosine kinases. In addition, PD 166285 inhibited PDGF- and EGF-stimulated receptor autophosphorylation in vascular smooth muscle cells (VSMCs) and A431 cells, respectively, and basic fibroblast growth factor-mediated tyrosine phosphorylation in Sf9 cells, with IC50 values of 6.5 nM, 1.6 microM and 97.3 nM, respectively, further establishing a tyrosine kinase mechanism of inhibition. The inhibition of PDGF receptor autophosphorylation in VSMCs by PD 166285 was long lasting and persisted for 4 days after a single 1-hr exposure followed by extensive washing. The PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen-activated protein kinase isoforms was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 166285 in VSMCs. The effects of PD 166285 were also demonstrated in functional assays of cell attachment, migration and proliferation, in which vascular cell adhesion to vitronectin, PDGF-directed chemotaxis and serum-stimulated cell growth were all potently inhibited with IC50 values of 80 yo 120 nM. Finally, PD 166285 uniquely demonstrated potent inhibition of phorbol ester-induced production of 92-kDa gelatinase A (MMP-9) in VSMC without affecting 72-kDa gelatinase B (MMP-2) as measured by gelatin zymography. These results highlight the biological characteristics of PD 166285 as a broadly active protein tyrosine kinase capable of potently inhibiting a number of kinase mediated cellular functions, including cell attachment, movement and replication. The potential therapeutic utility of this broadly acting inhibitor as an antiproliferative and antimigratory agent could extend to such diseases as cancer, atherosclerosis and restenosis, in which redundancies in protein kinase signaling pathways are known to exist.

The platelet-derived growth factors (PDGF A, B, C, and D) and their receptors (α-PDGFR and β-PDGFR) play an indispensible role in physiologic and pathologic conditions, including tumorigenesis. The transformative β-PDGFR is overexpressed and activated during prostate cancer progression, but the identification and functional significance of its complementary ligand have not been elucidated. This study examined potential oncogenic functions of β-PDGFR ligands PDGF B and PDGF D, using nonmalignant prostate epithelial cells engineered to overexpress these ligands. In our models, PDGF D induced cell migration and invasion more effectively than PDGF B in vitro. Importantly, PDGF D supported prostate epithelial cell tumorigenesis in vivo and showed increased tumor angiogenesis compared with PDGF B. Autocrine signaling analysis of the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways found PDGF D-specific activation of the c-jun-NH2-kinase (JNK) signaling cascade. Using short hairpin RNA and pharmacologic inhibitors, we showed that PDGFD-mediated phenotypic transformation is β-PDGFR and JNK dependent. Importantly, we made a novel finding of PDGF D-specific increase in the shedding and activation of the serine protease matriptase in prostate epithelial cells. Our study, for the first time to our knowledge, showed ligand-specific β-PDGFR signaling as well as PDGF D-specific regulation of matriptase activity and its spatial distribution through shedding. Taken together with our previous finding that matriptase is a proteolytic activator of PDGF D, this study provides a molecular insight into signal amplification of the proteolytic network and PDGF signaling loop during cancer progression.

Hypoxic-ischemic (H-I) injury to neonatal brains can cause a life-long neuronal deficit because of increased susceptibility in the neonatal period. Excitotoxicity due to overstimulation of the N-methyl-d-aspartate receptor (NMDAR) is assumed to be the basis of the injury. However, the ontogenic profile of the susceptibility does not directly correlate with the levels of NMDAR expression. Platelet-derived growth factor B-chain (PDGF-B) has been reported to protect neurons by suppressing the NMDA-evoked current and translocating the glutamate transporter to the cell membrane. Thus, we assessed the relationship between the susceptibility to H-I injury and the expression of PDGF-B in neonatal rat brain. PDGF-B infusion before and after an intrastriatal NMDA injection significantly reduced the size of the lesions in 7-day-old rats, when they are most susceptible and the neuronal expression of PDGF-B is low. Fourteen-day-old neonatal rats were found to be resistant to NMDA injury, even though NMDARs are expressed at high levels in the brain at this age. Inhibition of PDGF-B protein synthesis by antisense oligodeoxynucleotides increased the size of the NMDA-induced lesions up to 6-fold at postnatal day 14, when PDGF-B is expressed at high levels in neurons. These data suggest that PDGF-B is an important physiological modulator of NMDAR excitability in the developing brain, and that the balance between the expression of NMDAR and PDGF-B partly determines the ontogenic susceptibility to brain injury. Enhancement of the PDGF-B/receptor signal pathway might rescue neonatal brains at risk of H-I injury.