Platelet-derived Growth Factor Receptor Protein Research Articles

Abstract B066: PDGFR upregulation functions as a bypass mechanism contributing to FGFR inhibitor resistance in metastatic breast cancer

Abstract Breast cancer (BC) is the most diagnosed cancer in females in the United States and the second leading cause of cancer related deaths among women. The fibroblast growth factor receptor (FGFR) signaling pathway is frequently activated in BC, making it an attractive therapeutic target, especially in metastatic disease. However, clinical trials using FGFR inhibitors in BC have been disappointing in comparison to other cancer types treated with these therapies. As a result, BC patients with activated FGFR do not have a targeted therapeutic option. Failure of FGFR inhibitors is indicative of intrinsic or acquired resistance mechanisms in metastatic BC. Herein we demonstrate significant efficacy of the FGFR inhibitor, pemigatinib, in the 4T07 murine model of BC utilizing a tail vein model to induce pulmonary tumors. Despite this initial response, pemigatinib fails to eliminate minimal residual disease (MRD) and allows for accelerated tumor return after pemigatinib cessation. Characterizing the MRD by IHC following pemigatinib treatment shows that expression of platelet-derived growth factor receptor (PDGFR) is significantly increased on tumor cells. This rise in PDGFR expression following pemigatinib treatment increases the responsiveness of 4T07 cells to platelet-derived growth factor ligand indicating PDGFR may act as an alternate signaling pathway bypassing FGFR signaling. Mechanistically, previous studies indicate that PDGFR protein expression is transcriptionally repressed by restriction of enhancer positioning by the CTCF insulator. DNA methylation surrounding CTCF causes its dissociation enhancing protein expression of PDGFR. Along these lines cotreatment with a DNA methyl transferase inhibitor prevents the increase of PDGFR protein expression induced by pemigatinib. Overall, our study determined that a highly potent FGFR inhibitor can inhibit tumor growth in a metastatic site, but it fails to eliminate MRD giving rise to tumor recurrence. Upregulation of PDGFR may act as a bypass mechanism of resistance allowing for tumor cell survival during pemigatinib treatment. Finally, our findings suggest that dual inhibition of DNA methylation and FGFR could improve patient response in metastatic BC. Citation Format: Mitchell Ayers, Michael Wendt. PDGFR upregulation functions as a bypass mechanism contributing to FGFR inhibitor resistance in metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B066.

Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor–Mediated AXL Phosphorylation

Rationale & ObjectiveImmunoglobulin A nephropathy (IgAN) is a common glomerular disease, with mesangial cell proliferation as a major feature. There is no disease-specific treatment. Platelet-derived growth factor (PDGF) contributes to the pathogenesis of IgAN. To better understand its pathogenic mechanisms, we assessed PDGF-mediated AXL phosphorylation in human mesangial cells and kidney tissue biopsy specimens.Study DesignImmunostaining using human kidney biopsy specimens and in vitro studies using primary human mesangial cells.Setting & ParticipantsPhosphorylation of AXL was assessed in cultured mesangial cells and 10 kidney-biopsy specimens from 5 patients with IgAN, 3 with minimal change disease, 1 with membranous nephropathy, and 1 with mesangioproliferative glomerulonephritis (GN).PredictorGlomerular staining for phospho-AXL in kidney biopsy specimens of patients with mesangioproliferative diseases.OutcomesPhosphorylated AXL detected in biopsy tissues of patients with IgAN and mesangioproliferative GN and in cultured mesangial cells stimulated with PDGF.Analytic Approacht test, Mann-Whitney test, and analysis of variance were used to assess the significance of mesangial cell proliferative changes.ResultsImmunohistochemical staining revealed enhanced phosphorylation of glomerular AXL in IgAN and mesangioproliferative GN, but not in minimal change disease and membranous nephropathy. Confocal-microscopy immunofluorescence analysis indicated that mesangial cells rather than endothelial cells or podocytes expressed phospho-AXL. Kinomic profiling of primary mesangial cells treated with PDGF revealed activation of several protein-tyrosine kinases, including AXL. Immunoprecipitation experiments indicated association of AXL and PDGF receptor proteins. An AXL-specific inhibitor (bemcentinib) partially blocked PDGF-induced cellular proliferation and reduced phosphorylation of AXL and PDGF receptor and the downstream signals (AKT1 and ERK1/2).LimitationsSmall number of kidney biopsy specimens to correlate the activation of AXL with disease severity.ConclusionsPDGF-mediated signaling in mesangial cells involves transactivation of AXL. Finding appropriate inhibitors to block PDGF-mediated transactivation of AXL may provide new therapeutic options for mesangioproliferative kidney diseases such as IgAN.

Abstract CT009: A Phase Ib study assessing biomarker modulation in soft tissue sarcoma patients treated with olaratumab followed by olaratumab plus doxorubicin

Abstract Background: Olaratumab (olara), an IgG1 monoclonal antibody, blocks platelet-derived growth factor receptor (PDGFR) α activation. In a randomized Phase 2 study, olara plus doxorubicin (dox) improved overall survival in advanced soft tissue sarcoma (STS) patients (pts). The aim of this study (JGDM, NCT02783599) was to further evaluate potential biomarkers and the mechanism of action of olara in pts with STS. Methods: Pts with potentially resectable STS received olara monotherapy for one cycle followed by up to 6 cycles of olara plus dox (75 mg/m2 on Day 1). Olara was administered at 20 mg/kg (Cycles 1 and 2) and 15 mg/kg (Cycles 3-7) on Days 1 and 8 of a 21-day cycle. Co-primary objectives were enumeration of circulating tumor cells (CTCs) and PDGFR biomarker expression in tumor tissue pre- and post-olara monotherapy. PDGFR protein expression was analyzed by a validated immunohistochemistry (IHC) assay; Epic Sciences performed CTC enumeration. Secondary objectives included antitumor activity, safety, and pharmacokinetics (PK). Results: Of the 51 pts enrolled, 35 were evaluable for CTC enumeration and 43 were evaluable for PDGFR expression. Median number of CTCs detected before and after olara monotherapy were 1.0/mL (range: 0.1-9.2) and 0.1/mL (range: 0.1-18.7), respectively, in the analysis population. The proportion of pts with a decrease in CTC counts post-olara monotherapy in Cycle 1 was higher in pts with disease control (58%; 11 of 19 pts) compared with pts with no disease control (31%; 5 of 16 pts) after Cycle 2 (p=0.22). In the 43 pts evaluable for tumor tissue PDGFR analysis, IHC staining at baseline was positive in 31 (72.1%) pts for PDGFRα and 36 (83.7%) pts for PDGFRβ. Post-olara monotherapy samples showed similar rates of qualitative IHC expression for PDGFRα (n=30, 69.8%) and PDGFRβ (n=33, 76.7%). Qualitative IHC expression for either marker did not appear to correlate with clinical outcome in the analysis population. Clinically, overall response rate (CR+PR) was 11.8% (n=6), 27% of pts (n=14) had a decrease in size of target lesions, and 35.3% of pts (n=18) underwent tumor resection following systemic treatment. The safety and PK profiles of olara in combination with dox were consistent with previous reports. Conclusions: CTC enumeration in STS pts is feasible and disease control after Cycle 2 was associated with a statistically non-significant decrease in CTC following olara monotherapy in Cycle 1. Qualitative detection of PDGFRα in pre-treatment samples showed no association with clinical outcome and similar rates of qualitative detection in post-treatment samples. Further quantitative exploration of tissue biopsy results and correlations with exploratory findings in CTCs is ongoing. Citation Format: Javier Martin-Broto, Antonio Lopez Pousa, Andrew Brohl, Brian A. Van Tine, Benjamin Powers, Silvia Stacchiotti, Jean-Yves Blay, James S. Hu, Gerard J. Oakley, Hong Wang, Anna Szpurka, Donna E. Levy, Gary Mo, Matteo Ceccarelli, Robin Jones. A Phase Ib study assessing biomarker modulation in soft tissue sarcoma patients treated with olaratumab followed by olaratumab plus doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT009.

Analysis of genomic mutation and immunohistochemistry of platelet‐derived growth factor receptors in canine vascular tumours

We examined whether mutation of the platelet-derived growth factor receptor protein tyrosine kinase (PDGFR)-α and PDGFR-β genes contributes to their overexpression in canine vascular tumours. Genomic sequences of trans- or juxtamembrane regions of PDGFR-α and PDGFR-β were analysed with immunohistochemical staining and polymerase chain reaction-direct sequencing using DNA from paraffin-embedded neoplastic tissues of 27 hemangiosarcomas (HSAs) and 20 hemangiomas (HAs). Immunohistochemically, 75% of the HA cases were positive for PDGFR-α and almost most of the HA cases were negative for PDGFR-β. Of the HSA cases, 55.6% were negative for PDGFR-α and 63% were strongly positive for PDGFR-β. Among the HA cases, 1 missense mutation was detected in PDGFR-α exon 18 and 1 in PDGFR-β exon 17. Two HSA cases had missense mutations in exon 14 and 1 in exon 17 of PDGFR-β. Thus, genomic mutation of trans- or juxtamembrane regions of PDGFRs was not the main mechanism driving the activation of receptors in HSA and HA.

The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

Laminin α4-Null Mutant Mice Develop Chronic Kidney Disease with Persistent Overexpression of Platelet-Derived Growth Factor

Each extracellular matrix compartment in the kidney has a unique composition, with regional specificity in the expression of various laminin isoforms. Although null mutations in the majority of laminin chains lead to specific developmental abnormalities in the kidney, Lama4-/- mice have progressive glomerular and tubulointerstitial fibrosis. These mice have a significant increase in expression of platelet-derived growth factor (PDGF)-BB, PDGF-DD, and PDGF receptor beta in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to alpha4-containing laminins, but not other isoforms, results in down-regulation of PDGF receptor mRNA and protein, suggesting a direct effect of LN411/LN421 on vessel maturation. Given the known role of overexpression of PDGF-BB and PDGF-DD on glomerular and tubulointerstitial fibrosis, these data suggest that failure of laminin alpha4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model. Given the recent demonstration that individuals with laminin alpha4 mutations develop cardiomyopathy, these findings may be relevant to kidney disease in humans.

Protein expression of platelet-derived growth factor receptor correlates with malignant histology and PTEN with survival in childhood gliomas.

We previously showed that overexpression of epidermal growth factor receptor (EGFR) is associated with malignant grade in childhood glioma. The objective of this study was to determine whether protein expression of EGFR or platelet-derived growth factor receptor (PDGFR) and their active signaling pathways are related to malignant histology, progression of disease, and worse survival. Tissue microarrays were prepared from untreated tumors from 85 new glioma patients [22 high-grade gliomas (HGG) and 63 low-grade gliomas (LGG)] diagnosed at this institution from 1989 to 2004. Immunohistochemistry was used to assess total expression of EGFR, PDGFR beta, and PTEN and expression of phosphorylated EGFR, phosphorylated PDGFR alpha (p-PDGFR alpha), phosphorylated AKT, phosphorylated mitogen-activated protein kinase, and phosphorylated mammalian target of rapamycin. These results were correlated with clinicopathologic data, including extent of initial tumor resection, evidence of dissemination, tumor grade, proliferation index, and survival, as well as with Affymetrix gene expression profiles previously obtained from a subset of these tumors. High expression of p-PDGFR alpha, EGFR, PDGFR beta, and phosphorylated EGFR was seen in 85.7%, 80.0%, 78.9%, and 47.4% of HGG and 40.0%, 87.1%, 41.7%, and 30.6% of LGG, respectively. However, high expression of p-PDGFR alpha and PDGFR beta was the only significant association with malignant histology (P = 0.031 and 0.005, respectively); only the loss of PTEN expression was associated with worse overall survival. None of these targets, either alone or in combination, was significantly associated with progression-free survival in either LGG or HGG. High PDGFR protein expression is significantly associated with malignant histology in pediatric gliomas, but it does not represent an independent prognostic factor. Deficient PTEN expression is associated with worse overall survival in HGG.

Differential Behaviors of Atrial Versus Ventricular Fibroblasts

In various heart disease paradigms, atria show stronger fibrotic responses than ventricles. The possibility that atrial and ventricular fibroblasts respond differentially to pathological stimuli has not been examined. We compared various morphological, secretory, and proliferative response indexes of canine atrial versus ventricular fibroblasts. Cultured atrial fibroblasts showed faster cell surface area increases, distinct morphology at confluence, and greater alpha-smooth muscle actin expression than ventricular fibroblasts. Atrial fibroblast proliferation ([(3)H]thymidine incorporation) responses were consistently greater for a range of growth factors, including fetal bovine serum, platelet-derived growth factor (PDGF), basic fibroblast growth factor, angiotensin II, endothelin-1, and transforming growth factor-beta(1). Normal atrial tissue showed larger myofibroblast density compared with ventricular tissue, and the difference was exaggerated by congestive heart failure. Congestive heart failure atria showed larger fractions of fibroblasts in mitotic phases compared with ventricles and displayed enhanced gene expression of fibroblast-selective markers (collagen-1, collagen-3, fibronectin-1). Gene microarrays revealed 225 differentially expressed transcript probe sets between paired atrial and ventricular fibroblast samples, including extracellular matrix (eg, fibronectin, laminin, fibulin), cell signaling (PDGF, PDGF receptor, angiopoietin, vascular endothelial growth factor), structure (keratin), and metabolism (xanthine dehydrogenase) genes, identifying PDGF as a candidate contributor to atrial-ventricular fibroblast differences. PDGF receptor gene expression was greater in normal atrium compared with ventricle, and congestive heart failure differentially enhanced atrial versus ventricular PDGF and PDGF receptor gene expression. PDGF receptor protein expression and alpha-smooth muscle actin protein expression were enhanced in isolated congestive heart failure fibroblasts. The PDGF receptor tyrosine kinase inhibitor AG1295 eliminated fetal bovine serum- and transforming growth factor-beta(1)-stimulated atrial-ventricular fibroblast proliferative response differences. Atrial fibroblasts behave differently than ventricular fibroblasts over a range of in vitro and in vivo paradigms, with atrial fibroblasts showing enhanced reactivity that may explain greater atrial fibrotic responses. PDGF signaling is particularly important for atrium-selective fibroblast responses and may represent a novel target for arrhythmogenic atrial structural remodeling prevention.

Sunitinib: A VEGF and PDGF receptor protein kinase and angiogenesis inhibitor

Sunitinib (SU-11248, Sutent) inhibits at least eight receptor protein-tyrosine kinases including vascular endothelial growth factor receptors 1–3 (VEGFR1–VEGFR3), platelet-derived growth factor receptors (PDGFRα and PDGFRβ), stem cell factor receptor (Kit), Flt-3, and colony-stimulating factor-1 receptor (CSF-1R). VEGFR1 and VEGFR2 play key roles in vasculogenesis and angiogenesis. PDGFRβ, which is found in pericytes that surround capillary endothelial cells, plays a pivotal role in stabilizing the vascular endothelium. Sunitinib inhibits angiogenesis by diminishing signaling through VEGFR1, VEGFR2, and PDGFRβ. Renal cell cancers that have metastasized, or spread from the primary tumor, exhibit extensive vascularity, and sunitinib is approved for the treatment of these neoplasms. Activating Kit mutations occur in about 85% of gastrointestinal stromal tumors and activating PDGFRα mutations occur in about 5% of these tumors. Sunitinib is approved for the treatment of those tumors that are resistant to imatinib (STI-571, Gleevec), another Kit and PDGFRα protein-tyrosine kinase inhibitor. Both sunitinib and imatinib bind reversibly to the ATP binding site of their target kinases and thereby inhibit their catalytic activity.

Role of Platelet-derived Growth Factor and Vascular Endothelial Growth Factor in Obliterative Airway Disease

Platelet-derived growth factor (PDGF) is an important smooth muscle cell mitogen, and vascular endothelial growth factor (VEGF) is a known angiogenic and proinflammatory growth factor. We hypothesized that specific therapy aimed at these growth factors might inhibit the development of experimental obliterative airway disease (OAD). In fully mismatched rat tracheal allografts, we used imatinib and PTK/ZK, either alone or in combination, to block PDGF and VEGF receptor protein tyrosine kinase (RTK) action, respectively. Prophylaxis was initiated at the time of transplantation. Early treatment was commenced on Day 7 during the inflammatory phase and late treatment on Day 14 during the fibroproliferative phase of OAD. No immunosuppression was administered. Prophylaxis with either PTK/ZK or imatinib alone significantly reduced OAD, and combined prophylaxis completely prevented its development. Early treatment with PTK/ZK and imatinib also effectively reduced the development of OAD. Late treatment failed to show significant efficacy. Blocking VEGF RTK action with PTK/ZK reduced the activation of allograft blood vessels and the number of lymph vessels in the allograft airway wall, and significantly diminished allograft inflammation, whereas PDGF blockade with imatinib inhibited the growth of smooth muscle cells in the proliferating lesion. Combined prophylactic PDGF and VEGF RTK blockade completely prevents the development of OAD. Also, when early treatment with PTK/ZK and imatinib is commenced during the inflammatory phase of OAD development, it significantly attenuates the development of tracheal occlusion, suggesting that these drugs could potentially be used to treat bronchiolitis obliterans syndrome in its early phase.

Stimulation of Vascular Smooth Muscle Cell Migration by Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is a well known proinflammatory factor that influences the migration and proliferation of various cell types, predominantly monocytes and macrophages. Recent evidence suggests an important role for MIF in the progression of atherosclerosis and restenosis. For this reason, we studied the effect of MIF on platelet-derived growth factor-BB (PDGF-BB)-induced migration and PDGF receptor protein expression in vascular smooth muscle cells (VSMCs). Furthermore, the possibility of MIF influencing the migration of VSMCs was investigated. Our results show that short-term incubation of MIF is able to enhance PDGF-BB-induced migration. Long-term incubation decreases PDGF-BB-induced migration, but preserves a short-term stimulatory effect. These effects are not regulated at the level of PDGF receptor protein expression. MIF also acts as a chemoattractant for VSMCs, with a maximum response at 15 ng/ml. In contrast, the proliferation of VSMCs was unaffected by MIF. We conclude that MIF has a biphasic effect on VSMC migration. It remains unclear whether this effect is direct or involves the secretion of unidentified promigratory factors. Exogenous MIF does not stimulate VSMC proliferation; however, a role for MIF in proliferation cannot be fully ruled out. In view of the known key contributions of macrophage-derived MIF and VSMCs, the observed effects may well play a role in the progression of atherosclerosis and restenosis.

Expression of platelet-derived growth factor (PDGF) in the epididymis and analysis of the epididymal development in PDGF-A, PDGF-B, and PDGF receptor beta deficient mice.

The platelet-derived growth factor (PDGF) family of ligands and receptors play a pivotal role in the development of various organs. The critical importance of the PDGF-mediated signaling during embryonic development and adult physiology of the kidney and the common mesonephric origin of the epididymis and kidney prompted us to investigate the immunohistochemical localization of PDGF A- and B-chain and PDGF receptor (PDGFR) alpha- and beta-subunit in rat and mouse epididymis, the expression profiles of the corresponding mRNAs, and the consequences of a loss-of-function mutation at the PDGF-A, PDGF-B, and PDGFR-beta loci on mouse epididymis phenotypic appearance. Prenatally, PDGF-A and PDGFR-alpha immunohistochemical staining was seen in both species, whereas PDGF-B and PDGFR-beta were absent. The cellular localization of PDGF-A within the epithelium and the alpha-receptor in the mesenchyme in either mouse or rat before birth suggests that the PDGF-A/PDGFR-alpha system might be involved in the epididymal epithelial-mesenchymal interaction during the fetal period of life. Postnatally, PDGF A- and B-ligand and PDGFR alpha- and beta-subunit were confined in the epithelium. The identity of PDGF and PDGFR proteins were further confirmed by immunoblotting. In line with the immunohistochemical studies, PDGF-A and PDGFR-alpha mRNAs were seen by reverse transcription-polymerase chain reaction in rat and mouse tissue before birth, whereas PDGF-B and PDGFR-beta were almost not detectable. During the first days of life, PDGF-B and PDGFR-beta genes started to appear, and the overall trend in mRNA expression throughout postnatal development showed that the transcripts levels for PDGF-A, PDGF-B, PDGFR-beta, and PDGFR-alpha were constant with the only exception of a progressive decrease of PDGFR-alpha in adult rats. The PDGF-A null mutation strongly influenced the epididymal phenotype starting from puberty; only fetal PDGF-B and PDGFR-beta -/- mice were available, and no differences were seen in the epididymis of these animals, compared with wild-type littermates. Taken together, these data indicate that the PDGF system is highly expressed in the epididymis and suggest that PDGF could be involved in the maintenance of morphological structure and functional control of this organ.

Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR)-dependent signaling.

Angiogenesis and vascular cell proliferation are pivotal in physiological and pathological processes including atherogenesis, restenosis, wound healing, and cancer development. Here we show that mammalian target of rapamycin (mTOR) signaling plays a key role in hypoxia-triggered smooth muscle and endothelial proliferation and angiogenesis in vitro. Hypoxia significantly increased DNA synthesis and proliferative responses to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) in rat and human smooth muscle and endothelial cells. In an in vitro 3-dimensional model of angiogenesis, hypoxia increased PDGF- and FGF-stimulated sprout formation from rat and mouse aortas. Hypoxia did not modulate PDGF receptor mRNA, protein, or phosphorylation. PI3K activity was essential for cell proliferation under normoxic and hypoxic conditions. Activities of PI3K-downstream target PKB under hypoxia and normoxia were comparable. However, mTOR inhibition by rapamycin specifically abrogated hypoxia-mediated amplification of proliferation and angiogenesis, but was without effect on proliferation under normoxia. Accordingly, hypoxia-mediated amplification of proliferation was further augmented in mTOR-overexpressing endothelial cells. Thus, signaling via mTOR may represent a novel mechanism whereby hypoxia augments mitogen-stimulated vascular cell proliferation and angiogenesis.

New perspectives in PDGF receptor downregulation: the main role of phosphotyrosine phosphatases.

Uncontrolled activation of receptor tyrosine kinases (RTKs) is implicated in the proliferation of cancerous cells, and deficiencies in RTKs results in pathological conditions such as developmental abnormalities and immunodeficiencies. Tight regulation of RTK cascades is therefore critical for eliciting an appropriate type and level of response to external stimuli. The aim of this work is to compare different RTK downregulation mechanisms, such as ligandinduced internalisation, ubiquitin-mediated proteolysis and dephosphorylation by protein phosphotyrosine phosphatase (PTPs). We choose platelet-derived growth factor receptor (PDGF-r) in NIH3T3 cells as a model of RTK. Our data suggest that PDGF-r internalisation could be mainly considered as a positive signaling system, as it is involved in MAPK activation rather than a downregulation of the mitotic signal. Inhibition of receptor ubiquitination does not result in regulation of PDGF-r tyrosine phosphorylation and does not lead to variation of intracellular signalling pathways. The overall PDGF-r protein degradation upon PDGF stimulation does not exceed 30-40% of the total receptor; thus the receptor remains functionally active for further stimulation. On the contrary, PTP-dependent dephosphorylation of the activated receptors appears to play a crucial role. In fact, inhibition of PTP upon PDGF stimulation results in upregulation of receptor phosphorylation level, of PI3K recruitment and activation and of cell cycle rate. On the contrary, PTP-dependent dephosphorylation does not affect the endosomic pool of activated receptor. Furthermore, we demonstrate that PDGF-r downregulation by means of PTP dephosphorylation is important for both short term (2 hours) and long-lasting (up to 8 hours) PDGF-r activation. Herein we propose a revisited model of PDGF-r downregulation in which PTPs dephosphorylation retains a major role, conferring on receptor internalisation a signal transduction function.

Expression of platelet-derived growth factor-A (PDGF-A), PDGF-B, and PDGF receptor-alpha and -beta during human testicular development and disease.

Platelet-derived growth factor (PDGF) plays a critical role in regulating the development and functional control of various tissues and has been implicated in the pathogenesis of serious diseases, including cancer and atherosclerosis. Given the emerging role of PDGF in the development and function of male gonads, we compared the expression profiles of the mRNAs of the PDGF A- and B-chains and of the PDGF receptor (PDGFR) alpha- and beta-subunits in fetal and adult human testis. The immunohistochemical localization of the corresponding proteins in fetal, adult, and diseased human testicular tissues was also analyzed. PDGFs and PDGFRs mRNAs were readily detected by both Northern analysis and RT-PCR. The transcript levels were higher during 16-20 wk gestation, significantly lower at 24-28 wk, and increased in the adult. An identical pattern of protein expression was confirmed by immunohistochemistry, although the cellular localization of the PDGF system changes during postnatal development, concomitantly with the progression of spermatogenesis. In the testicular samples from patients affected by either complete aplasia of germ cells or various grades of spermatogenic arrest, the immunohistochemical localization of PDGFs and PDGFRs was different from normal, confirming a close connection between germ cells and PDGF system distribution. These results indicate that PDGF, through complex interactions, could play a leading role in ontogenesis and testicular pathophysiology in humans. Finally, the expression of PDGF ligands and receptor proteins in Leydig cell tumors suggests a relationship of the PDGF system to tumorigenesis or tumor progression in this testicular neoplasm.

Agonist-specific differential changes of cellular signal transduction pathways in senescent human diploid fibroblasts

Changes in the signal transduction efficiency of senescent cells led us to compare the signaling events induced by two mitogenic agonists, platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) in presenescent and senescent or near-senescent human diploid fibroblasts. When the changes in intracellular [Ca 2+] i were analyzed, both PDGF and LPA generated a rhythmic increase in [Ca 2+] i in presenescent cells. The frequency of calcium response was reduced and desensitized in PDGF-stimulated senescent cells, while response to a LPA-induced calcium signal was also reduced in frequency, though its magnitude was unaltered. PDGF treatment increased the fibrous actin (F-actin) level in presenescent cells but not in senescent cells in contrast to a reduced but visible increase in F-actin in LPA-treated senescent cells. The effect of PDGF on phospholipase D (PLD) activation was also reduced significantly, as a ca. 60–80% reduction of PLD activity was observed in PDGF-stimulated cells but only a little reduction in LPA-induced cells. Agonist-specific differential changes of cellular signaling events caused a differential effect on DNA synthesis after growth factor stimulation. We observed a dramatic (80–90%) reduction of [ 3H]thymidine incorporation into DNA in the PDGF-stimulated near-senescent cells. LPA resulted in a 2–3-fold increase in thymidine incorporation even in the near-senescent cells. These differences in the responses of senescent or near-senescent cells to PDGF- and LPA-stimulation raised questions about the differential changes of the respective signaling apparatuses induced by aging. Since PDGF signaling event was affected greatly by aging, we further examined the protein contents involved in PDGF signal transduction pathway. PDGF receptor (PDGFR), protein kinase C-α (PKC-α), phospholipase C-γ1 (PLC-γ1), and PLD1 were examined by Western blot analysis. The protein levels of PKC-α and PLC-γ1 were unchanged, but those of PLD1 and PDGFR were reduced with age. The reduced content of PDGFR protein may be one of the important contributors to the failure of PDGF-stimulated signal transduction in human senescent fibroblasts. Our results strongly suggest that age-dependent agonist-specific changes in signaling events might be in charge of the functional deterioration of senescent cells through imbalance of signal responses.

Mechanical stretch augments PDGF receptor beta expression and protein tyrosine phosphorylation in pulmonary artery tissue and smooth muscle cells.

With regard to the mechanotransduction mechanisms of vasculature involved in hypertensive diseases, we aimed to identify tyrosine-phosphorylated proteins in pulmonary artery that responded to mechanical stress. Mechanical stretch simultaneously augmented protein-tyrosine phosphorylation in p55, p95, p105, p115, p130, p165, p180 in pulmonary artery tissue and pulmonary artery-derived smooth muscle cells (PASMC), whereas p115 and p55 were preferentially phosphorylated by the stretch in endothelial cells (PAEC). A series of experiments designed to characterize these proteins indicated that p115 and p180 were focal adhesion kinase (FAK) and platelet-derived growth factor receptor beta (PDGF-Rbeta), respectively, and that stretch augmented the surface-expression of PDGF-Rbeta in PASMC but not in PAEC. Moreover, a significant increase in the steady-state mRNA level for PDGF-Rbeta was observed in the pulmonary artery of rats with monocrotaline-induced pulmonary hypertension, where the artery should be overstretched due to increasing pulmonary arterial blood pressure. These results suggest that stretch-induced overexpression of cell-surface PDGF-Rbeta as well as augmentation of yrosine phosphorylation of proteins including FAK in PASMC might be involved in the mechanotransduction of pulmonary artery.

Small-molecule control of insulin and PDGF receptor signaling and the role of membrane attachment

Background: Receptor tyrosine kinases (RTKs) regulate the proliferation, differentiation and metabolism of cells, and play key roles in tissue repair, tumorigenesis and development. To facilitate the study of RTKs, we have made conditional alleles that encode monomeric forms of the normally heterotetrameric insulin receptor and monomeric platelet-derived growth factor (PDGF) β receptors fused to the FK506-binding protein 12 (FKBP12). The chimeric receptors can be induced to undergo dimerization or oligomerization by a small synthetic molecule called FK1012, and the consequences were studied in cells and embryonic tissues.Results: When equipped with an amino-terminal plasma membrane localization sequence and expressed in HEK293 cells, these chimeric receptors could signal to downstream targets as indicated by the FK1012-dependent activation of p70 S6 kinase (p70S6k) and mitogen-activated protein (MAP) kinase. In Xenopus embryos, the engineered PDGF receptor protein induced the formation of mesoderm from animal-pole explants in an FK1012-dependent manner. A cytosolic variant of the protein underwent efficient transphosphorylation, yet failed to activate appreciably either p70S6k or MAP kinase following treatment with FK1012. These results provide evidence of a requirement for membrane localization of RTKs, consistent with current models of RTK signaling.Conclusion: We have developed an approach using the small molecule FK1012 to conditionally activate chimeric proteins containing FKBP fused to the insulin receptor or to the PDGF β receptor. Using this system, we were able to induce mesoderm formation in Xenopus animal-cap tissue and to demonstrate that membrane localization is required for RTK signaling in transfected cells. This system should allow the further dissection of RTK-mediated pathways.

Inhibition of platelet-derived growth factor receptor tyrosine kinase by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) is known to suppress platelet-derived growth factor (PDGF)-stimulated proliferation of rat cultured vascular smooth muscle cells. The present study examined whether ANP inhibits the PDGF receptor (PDGFR) tyrosine kinase activation, an initial event for PDGF cellular signaling. ANP reduced the in vivo tyrosine phosphorylation of PDGFR stimulated by PDGF in a dose-dependent manner. This effect was not due to the reduction in PDGFR protein as detected by immunoblot analysis. 8-Bromo-cyclic GMP, a membrane-permeable 3',5'-cyclic monophosphate (cGMP) derivative, mimicked the action of ANP. HS-142-1, an antagonist for guanylate cyclase A (GC-A) and B, co-incubated with ANP, restored the PDGF-induced PDGFR autophosphorylation. The effect of ANP was also observed in the presence of a protein tyrosine phosphatase inhibitor, sodium orthovanadate. To confirm that ANP exerts its action by inhibiting protein tyrosine kinase (PTK), an in vitro kinase assay was performed. Cyclic GMP inhibited PTK activity of PDGFR partially purified by lectin affinity chromatography. In contrast, PTK activity in immobilized PDGFR immunocomplexes was not inhibited by cGMP. However, exogenous cGMP dependent protein kinase (PKG) reduced the PTK activity in the presence of cGMP. These results demonstrate that ANP suppresses PDGFR PTK through GC-A probably by activating PKG. This may be an important mechanism by which ANP exerts its anti-proliferative action antagonizing PDGF.

PDGF receptor protein tyrosine kinase expression in the balloon-injured rat carotid artery.

Platelet-derived growth factor (PDGF) receptor gene expression has previously been demonstrated in balloon-injured rat carotid arteries to be regulated during repair of carotid injury. In the present study we showed that PDGF receptor protein expression and phosphorylation are changed over time after carotid artery injury. In control and 2-day-postinjury vessels, expression of PDGF alpha receptor protein was readily detectable, whereas PDGF beta receptor expression appeared very low. Between 2 and 7 days postinjury, a time interval previously shown to correspond with smooth muscle cell migration followed by the appearance of a neointima, PDGF alpha receptor expression had increased only slightly, to roughly 35% above control levels, and was maximal by day 7 postinjury, whereas PDGF beta receptor expression had doubled. From 7 to 14 days after carotid injury, intimal area was greatly increased and was associated with a further increase in PDGF beta receptor protein expression and receptor phosphorylation to a maximum between days 10 and 12. In contrast, PDGF alpha receptor expression had decreased slightly during this time interval. Moreover, phosphorylation of PDGF alpha receptors was barely detectable and did not change over the time course of injury. From 14 to 28 days after injury, intimal area was increased only slightly, whereas PDGF beta receptor protein and phosphorylation levels had diminished to roughly half of the 10-day injury values. In addition, the increase in PDGF beta receptor protein expression and tyrosine phosphorylation observed over the time of injury were also associated with a corresponding increase in the association of phosphatidylinositol 3' kinase (PI-3 kinase) with phosphorylated PDGF beta receptors. These findings show that balloon injury to rat carotid arteries results in temporally related changes in the expression of PDGF receptors and their state of tyrosine phosphorylation. Furthermore, tyrosine phosphorylation of PDGF beta receptors in the balloon-injured rat carotid artery in vivo resulted in the association of PI-3 kinase. These are important new findings, which add to our knowledge concerning the role and activity of PDGF receptors in the formation of a neointima.