Platelet-derived growth factor induces fetal wound fibrosis

The fetal response to cutaneous injury differs markedly from that of the adult, proceeding with only minimal inflammation, minimal fibroblast proliferation, and only essential collagen deposition. Although the sequence of events in adult wound healing is well defined and thought to be controlled in part by potent polypeptide cytokines, relatively sparse information exists regarding growth factor involvement in fetal wound repair. Thus, the authors sought to examine the effect of platelet-derived growth factor (PDGF), a putative adult wound healing regulator, on the cellular and extracellular matrix events at a fetal wound site. SILASTIC wound implants containing 0, 1.0, 5.0, or 10.0 ng of human PDGF were placed subcutaneously on the backs of 24-day-gestation fetal rabbits (full term, 31 days) and then harvested after either 1, 3, or 5 days in utero. The specimens underwent standard histological processing and were evaluated in a blinded fashion. Compared with controls, PDGF-treated implants had a marked increase in acute inflammation, fibroblast recruitment, and collagen and hyaluronic acid deposition; these differences appeared to be largely time- and PDGF dose-dependent. Thus, the fetal system is responsive to an adult wound healing mediator, and these data suggest that fetal repair proceeds in the absence of PDGF.     

Tyrosine phosphorylation and src family kinases control keratinocyte cell-cell adhesion

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that beta-catenin, gamma-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of alpha-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of alpha-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of beta- and gamma-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn-/- mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of beta-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.     

Small molecule inhibitors of the platelet-derived growth factor receptor, the fibroblast growth factor receptor, and Src family tyrosine kinases

The inhibition of tyrosine kinases involved in growth factor signal transduction pathways represents an attractive strategy for controlling aberrant cellular growth. Over the last 4-5 years, there have been numerous reports on the discovery of small molecule inhibitors for potential therapeutic applications to a number of proliferative diseases, principally cancer and restenosis, where the over-expression of certain tyrosine kinases has been demonstrated. These include, amongst others, the platelet-derived growth factor receptor, the fibroblast growth factor receptor, and the nonreceptor c-Src tyrosine kinase. This review compiles published reports and patent filings from 1995 to mid-1997 that include data directly related to inhibition of the platelet-derived growth factor receptor, fibroblast growth factor receptor, and Src family tyrosine kinases. Potential clinical applications for selected classes of tyrosine kinase inhibitors reviewed herein will likely depend on the demonstration of meaningful activity in a variety of therapeutic targets in animal models.     

Platelet-derived endothelial cell growth factor thymidine phosphorylase in tumour growth and response to therapy

Angiogenesis plays an important role in the growth and metastasis of solid tumours. Platelet-derived endothelial cell growth factor (PD-ECGF) is known to be chemotactic for endothelial cells in vitro and angiogenic in vivo. It is also known as gliostatin, a factor promoting neuronal survival, and thymidine phosphorylase (dThdPase), which catalyses the reversible phosphorylation of thymidine to thymine and 2-deoxyribose-1-phosphate. This enzymatic activity is critical for angiogenic activity. PD-ECGF protein is highly expressed in tumours compared with most normal tissues and has been correlated with tumour growth, invasion and metastasis in clinical studies. In addition, dThdPase activity (by inference PD-ECGF) has been found to be a major determinant of the toxicity of 5-fluorouracil and its prodrugs, which are extensively studied clinically as anti-cancer agents. This review attempts to summarize recent gains in understanding the nature, location and action of PD-ECGF and its specific relevance to tumour biology.     

Platelet-derived growth factor (PDGF) stimulates the association of SH2-Bbeta with PDGF receptor and phosphorylation of SH2-Bbeta

We recently identified SH2-Bbeta as a JAK2-binding protein and substrate involved in the signaling of receptors for growth hormone and interferon-gamma. In this work, we report that SH2-Bbeta also functions as a signaling molecule for platelet-derived growth factor (PDGF). SH2-Bbeta fused to glutathione S-transferase (GST) bound PDGF receptor (PDGFR) from PDGF-treated but not control cells. GST fusion protein containing only the SH2 domain of SH2-Bbeta also bound PDGFR from PDGF-treated cells. An Arg to Glu mutation within the FLVRQS motif in the SH2 domain of SH2-Bbeta inhibited GST-SH2-Bbeta binding to tyrosyl-phosphorylated PDGFR. The N-terminal truncated SH2-Bbeta containing the entire SH2 domain interacted directly with tyrosyl-phosphorylated PDGFR from PDGF-treated cells but not unphosphorylated PDGFR from control cells in a Far Western assay. These results suggest that the SH2 domain of SH2-Bbeta is necessary and sufficient to mediate the interaction between SH2-Bbeta and PDGFR. PDGF stimulated coimmunoprecipitation of endogenous SH2-Bbeta with endogenous PDGFR in both 3T3-F442A and NIH3T3 cells. PDGF stimulated the rapid and transient phosphorylation of SH2-Bbeta on tyrosines and most likely on serines and/or threonines. Similarly, epidermal growth factor stimulated the phosphorylation of SH2-Bbeta; however, phosphorylation appears to be predominantly on serines and/or threonines. In response to PDGF, SH2-Bbeta associated with multiple tyrosyl-phosphorylated proteins, at least one of which (designated p84) does not bind to PDGFR. Taken together, these data strongly argue that, in response to PDGF, SH2-Bbeta directly interacts with PDGFR and is phosphorylated on tyrosine and most likely on serines and/or threonines, and acts as a signaling protein for PDGFR.     

Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae

The ability of a peptide hormone to affect many different intracellular targets is thought to be possible because of the modular organization of signal transducing molecules in the cell. Evidence for the presence of signaling modules in metazoan cells, however, is incomplete. Herein we show, with morphology and cell fractionation, that all the components of a mitogen-activated protein kinase pathway are concentrated in caveolae of unstimulated human fibroblasts. Addition of platelet-derived growth factor to either the intact cell or caveolae isolated from these cells stimulates tyrosine phosphorylation and activates mitogen-activated protein kinases in caveolae. The molecular machinery for kinase activation, therefore, is preorganized at the cell surface of quiescent cells.     

A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development

Two live attenuated single-deletion mutant simian immunodeficiency virus (SIV) constructs, SIV239Deltanef and SIVPBj6.6Deltanef, were tested for their abilities to stimulate protective immunity in macaques. During the immunization period the animals were examined for specific immune responses and virus growth. Each construct generated high levels of specific immunity in all of the immunized animals. The SIV239Deltanef construct was found to grow to high levels in all immunized animals, with some animals remaining positive for virus isolation and plasma RNA throughout the immunization period. The SIVPBj6.6Deltanef was effectively controlled by all of the immunized animals, with virus mostly isolated only during the first few months following immunization and plasma RNA never detected. Following an extended period of immunization of over 80 weeks, the animals were challenged with a pathogenic simian-human immunodeficiency virus (SHIV) isolate, SIV89. 6PD, by intravenous injection. All of the SIV239Deltanef-immunized animals became infected with the SHIV isolate; two of five animals eventually controlled the challenge and three of five animals, which failed to check the immunizing virus, progressed to disease state before the unvaccinated controls. One of five animals immunized with SIVPBj6.6Deltanef totally resisted infection by the challenge virus, while three others limited its growth and the remaining animal became persistently infected and eventually died of a pulmonary thrombus. These data indicate that vaccination with attenuated SIV can protect macaques from disease and in some cases from infection by a divergent SHIV. However, if animals are unable to control the immunizing virus, potential damage that can accelerate the disease course of a pathogenic challenge virus may occur.     

Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture

The effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.     

Evidence for phosphorylation of CTP:phosphocholine cytidylyltransferase by multiple proline-directed protein kinases

Reversible phosphorylation of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of phosphatidylcholine biosynthesis, is thought to play a role in regulating its activity. In the present study, the hypothesis that proline-directed kinases play a major role in phosphorylating cytidylyltransferase is substantiated using a c-Ha-ras-transfected clone of the human keratinocyte cell line HaCaT. Cellular extracts from epidermal growth factor-stimulated HaCaT cells and from ras-transfected HaCaT cells phosphorylated cytidylyltransferase much stronger as compared with extracts from quiescent HaCaT cells. The tryptic phosphopeptide pattern of cytidylyltransferase phosphorylated by cell-free extracts from ras-transfected HaCaT cells was similar compared with the patterns of cytidylyltransferase phosphorylated by p44mpkmitogen-activated protein kinase and p34cdc2 kinase in vitro, whereas in the case of casein kinase II the pattern was different. Furthermore, in c-Ha-ras-transfected HaCaT cells the in vivo phosphorylation state of cytidylyltransferase was 2-fold higher as compared with untransfected HaCaT cells. This higher phosphorylation of cytidylyltransferase in the ras-transfected clone was reduced to a level below the phosphorylation of cytidylyltransferase in untransfected cells, using olomoucine, a specific inhibitor of proline-directed kinases. The reduced phosphorylation of cytidylyltransferase in olomoucine-treated cells correlated with an enhanced stimulation of enzyme activity by oleic acid.     

Platelet-derived growth factor reverses the effects induced by NSAIDs on ulcer healing

BACKGROUND: It is known that non-steroidal anti-inflammatory drug (NSAID) use delays the healing of peptic ulcers and that growth factors play an important role in the ulcer healing process. AIM: To evaluate the effect of platelet-derived growth factor (PDGF) in healing chronic gastric ulcers in rats treated with NSAIDs. METHODS: Chronic gastric ulcers were induced with acetic acid in male Wistar rats and then treated with either aspirin (100 mg/kg/day), indomethacin (2 mg/kg/day), PDGF-BB (0.1 nM/kg/day) or combinations. Gastric secretion and ulcer size, wound contraction, mucosal regeneration and cell proliferation were assessed in histological specimens. RESULTS: Both aspirin and indomethacin delayed the healing rate of gastric ulcers and reduced ulcer contraction, mucosal regeneration and cell proliferation. All these effects were completely reversed by oral treatment with PDGF-BB without affecting gastric acid secretion. CONCLUSION: Oral administration of PDGF accelerates ulcer healing and reverses the effects induced by NSAIDs on ulcer healing without affecting gastric secretion.     

Insulin-like growth factor receptors and binding proteins

The insulin-like growth factor receptors are integral membrane proteins and demonstrate separate, but important effects on the regulation of cellular processes. The IGF-I receptor signals multiple cascades via its inherent tyrosine kinase activity. The IGF-II/M-6-P receptor on the other hand is primarily involved in targeting of enzymes to various subcellular compartments. In contrast, the insulin-like binding proteins are secreted by the cells and accumulate in the extracellular matrix or on the external surface of the cell. They are also involved in regulating cellular processes more indirectly. They modulate the interactions of the IGFs with their receptors, and in addition, may have some IGF-independent effects probably by direct interaction with integrin and other cell membrane receptor proteins. The recent studies, as outlined in this review, strongly suggest an important, if not essential role for the IGF system in normal physiology and disease states. The challenge now is to define the mechanisms involved in these effects. More studies are required to fully understand the post-receptor mechanism involved in IGF-I receptor signal transduction and the mechanisms whereby the IGFBPs exert their interesting effects. Understanding these mechanisms will enable investigators to create new therapeutic modalities for diseases that are affected by the IGF system.     

Growth hormone stimulates tyrosine phosphorylation of JAK2 and STAT5, but not insulin receptor substrate-1 or SHC proteins in liver and skeletal muscle of normal rats in vivo

GH has been shown to stimulate tyrosine phosphorylation of JAK2, several STAT proteins, insulin receptor substrate-1 (IRS-1), and SHC proteins in cultured cells. The goal of this study was to determine GH effects on protein tyrosine phosphorylation in liver and skeletal muscle of normal rats in vivo. Nonfasted male Sprague-Dawley rats (225-250 g) were injected with GH iv, and tissues were obtained after 5, 15, 30, or 60 min. At a maximally effective GH dose (1.5 mg/kg body weight), phosphotyrosine antibody immunoblots demonstrated marked stimulation of the tyrosine phosphorylation of JAK2 (maximal at 5 min) and a 95,000 Mr protein (maximal at 15 min) in both liver and skeletal muscle. The 95,000 Mr protein was recognized and immunodepleted by STAT5 antibody, but not by other STAT protein antibodies. Although basal tyrosine phosphorylation of IRS-1 and SHC was evident, GH did not stimulate tyrosine phosphorylation of either of these proteins in liver or skeletal muscle. In conclusion, GH stimulates the tyrosine phosphorylation of JAK2 and STAT5, but not IRS-1, SHC, or other STAT proteins in liver and skeletal muscle of normal rats. These results differ from findings in cultured cells and support the concept that selectivity for tyrosine kinase substrates is an important determinant of postreceptor signaling specificity in vivo.     

Platelet-derived growth factor stimulates the release of protein kinase A from the cell membrane

The mitogenic action of growth factors involves the stimulation of intracellular protein kinases. In this report we have characterized the major protein kinase released from Balb/c 3T3 and normal rat kidney plasma membranes by the action of platelet-derived growth factor (PDGF). PDGF appears to stimulate the release of approximately 10 proteins, at least one of which is a kinase capable of phosphorylating proteins on Ser or Thr (as determined by the lability of the phosphate to alkali treatment). More than 90% of the Ser/Thr kinase activity was inhibited by PKI5-22, a specific peptide inhibitor of the cAMP-dependent protein kinase (PKA). We used immunoblotting to confirm that the kinase released in response to PDGF was PKA. cAMP also stimulated the release of PKA, and the set of protein substrates phosphorylated was similar following PDGF or cAMP stimulation. Interestingly, in the presence of a cAMP analogue ((Rp)-cAMPS), cAMP could not induce dissociation of PKA from the membranes, whereas stimulation by PDGF increased the level of PKA activation. Furthermore, unlike Swiss 3T3 cells, neither Balb/c 3T3 fibroblasts nor normal rat kidney cells accumulate cAMP in response to PDGF, yet the level of PKA in the cytosol of these intact cells increases in response to PDGF. Thus, it appears as though PDGF activation of the membrane-associated form of the PKA holoenzyme occurs by a mechanism independent of an elevation in cAMP levels.     

Mechanism of inhibition of eosinophil activation by transforming growth factor-beta. Inhibition of Lyn, MAP, Jak2 kinases and STAT1 nuclear factor

The activation of eosinophils by IL-5 plays a crucial role in the pathogenesis of allergic and parasitic disorders. IL-5 has recently been shown to activate Lyn and Jak2 tyrosine kinases, MAP kinases, and STAT1 nuclear factor. We have previously reported that TGF-beta blocks the IL-5-induced activation of eosinophils. In this study, we investigated the effect of TGF-beta on the IL-5-induced signaling molecules in eosinophils. Purified eosinophils from mildly allergic patients were preincubated with TGF-beta and then stimulated with IL-5. The cell lysates were then immunoprecipitated and blotted with antiphosphotyrosine Abs. The activity of the kinases was further studied in the immune-complex kinase assay. We found that TGF-beta inhibited the tyrosine phosphorylation of multiple proteins in eosinophils. The identity of some of the proteins was established by immunoprecipitation. We found that TGF-beta inhibited tyrosine phosphorylation of Lyn, Jak2, and a 44-kDa MAP kinase. In further experiments, it blocked the activation of the above kinases as determined by immune-complex kinase assay. TGF-beta also inhibited phosphorylation of the STAT1 (p91) nuclear protein in eosinophils. We believe that the inhibition of Lyn, Jak2, MAP kinase, and the STAT1 nuclear protein may underlie the inhibitory activity of TGF-beta on eosinophils.     

Inhibition of growth of primary human tumour cell cultures by a 4-anilinoquinazoline inhibitor of the epidermal growth factor receptor family of tyrosine kinases

The epidermal growth factor receptor (EGFR) is thought to mediate the action of the mitogens EGF and tumour growth factor-alpha (TGF-alpha) in a variety of cancers, including those of the lung, breast and ovary. A number of new selective inhibitors of EGFR tyrosine kinase have now been developed as potential new antitumour agents. We used a potent inhibitor of this tyrosine kinase, 6-amino-4-[(3-bromophenyl)amino]-7-(methylamino)quinazoline (SN 25531; PD 156273), to determine the responses of primary cultures derived from patients with cancer of the lung, ovary, breast, cervix and endometrium. Cells were cultured in 96-well plates and proliferation assessed by incorporation of 3H-thymidine. Measured growth inhibitory concentrations IC50 values) varied from 1 nM to 14 microM with a 1000-fold differential between sensitive and resistant cultures. Results were compared with rates of proliferation, estimated using a paclitaxel-based method. We also measured the IC50 values for the tyrosine kinase inhibitor using a number of established human cell lines, and compared them with EGFR content using fluorescent antibody staining and flow cytometry. The presence of EGFR was found to be necessary, but not sufficient, for in vitro response. Only a small number of cell lines (3 of 7 for lung, 1 of 7 for ovarian, 2 of 3 squamous cell and 0 of 12 for melanoma) were sensitive to the tyrosine kinase inhibitor. In contrast, 40 of the 50 primary cultures (including 14 of 15 lung cancer samples and 14 of 19 ovarian cancer samples) were sensitive.     

Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins

Physical forces play a fundamental role in the regulation of cell function in many tissues, but little is known about how cells are able to sense mechanical loads and realize signal transduction. Adhesion receptors like integrins are candidates for mechanotransducers. We used a magnetic drag force device to apply forces on integrin receptors in an osteoblastic cell line and studied the effect on tyrosine phosphorylation as a biochemical event in signal transduction. Mechanical stressing of both the beta1 and the alpha2 integrin subunit induced an enhanced tyrosine phosphorylation of proteins compared with integrin clustering. Application of cyclic forces with a frequency of 1 Hz was more effective than a continuous stress. Using Triton X-100 for cell extraction, we found that tyrosine-phosphorylated proteins became physically anchored to the cytoskeleton due to mechanical integrin loading. This cytoskeletal linkage was dependent on intracellular calcium. To see if mechanical integrin stressing induced further downstream signaling, we analyzed the activation of mitogen-activated protein (MAP) kinases and found an increased phosphorylation of MAP kinases due to mechanical stress. We conclude that integrins sense physical forces that control gene expression by activation of the MAP kinase pathway. The cytoskeleton may play a key role in the physical anchorage of activated signaling molecules, which enables the switch of physical forces to biochemical signaling events.     

Granulocyte-macrophage colony-stimulating factor induces a unique set of STAT factors in murine dendritic cells

Cytokine-mediated signaling pathways were studied in mouse dendritic cells (DC) by analysis of the activation pattern of STAT factors. Electrophoretic mobility shift assays were performed to detect STAT isoform-specific complexes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) simultaneously induced complexes containing STAT1, STAT3, STAT5A, STAT5B and STAT6. In non-DC, a similar broad activation pattern of STAT factors by GM-CSF or other cytokines has not been observed so far. By comparison, in peritoneal macrophages, GM-CSF induced a complex with the properties of a truncated form of STAT5. Other cytokines tested on DC either failed to induce STAT factors [interleukin (IL)-1 beta, IL-2, IL-15], or activated the same STAT factors as observed in peritoneal macrophages (IL-4, IFN-gamma). Our results implicate a specific effect of GM-CSF on STAT signaling in DC which might account for the cell type-specific effect of this cytokine on development and function.