Crosstalk between G protein-coupled receptors and tyrosine kinase signaling: Src take centre stage.

Although the role of G protein-coupled receptors in the regulation of metabolic, secretory and contractile responses is well established, they have only recently been recognized as important mediators of cellular growth and differentiation. G protein-coupled signaling pathways had been previously thought to be totally independent of the tyrosine kinase receptor pathway. It was previously believed that molecular switches responsible for growth factor tyrosine kinase receptor signaling and G protein-coupled signaling were divided into a distinct sets of protein families. Recent evidence has demonstrated, however, that G protein-coupled receptors can crosstalk to tyrosine kinase signaling. In the past few years several groups have found that G protein-coupled receptors utilize non-receptor tyrosine kinases, mostly that of Src family, and some adapter proteins, to regulate tyrosine kinase cascades in cells.

[Regulation of cellular differentiation and cellular function by phosphatidylinositol 3-kinase]. / Regulacja róznicowania komórkowego i funkcji komórkowych poprzez kinaze 3-fosfatydyloinozytolu.

Phosphatidylinositol 3-kinases (PI3Ks) generate lipids that are implicated in receptor-stimulated signalling and in the regulation of cell growth/differentiation and cellular function. Several pathways have been recently identified and this article summarizes current knowledge about them. Depending on cell type the PI3K pathway has been involved in positive or negative regulation of differentiation. Products of PI3Ks and other signalling intermediates are shared between the G protein-coupled receptors and receptor tyrosine kinases, suggesting that control of differentiation and growth is equally dependent on these two distinct classes of receptors. Thus, the role of PI3Ks in the regulation of differentiation is clearly a very complex process implicating integration between different receptor systems and cell type-specific responses.

Requirement of phosphatidylinositol 3-kinase activity for bradykinin stimulation of NF-kappaB activation in cultured human epithelial cells.

The signaling mechanisms utilized by bradykinin (BK) to activate the transcription factor nuclear factor kappaB (NF-kappaB) are poorly defined. We previously demonstrated that BK-stimulated NF-kappaB activation requires the small GTPase RhoA. We present evidence that BK-induced NF-kappaB activation both activates and requires phosphatidylinositol 3-kinase (PI 3-kinase) in A549 human epithelial cells. Pre-treatment with the PI 3-kinase-specific inhibitors, wortmannin, and LY294002 effectively blocked BK-induced PI 3-kinase activity. Wortmannin and LY294002 also abolished BK-induced NF-kappaB activation, as did transient transfection with a dominant negative mutant of the p85 subunit. BK-stimulated PI 3-kinase activity and NF-kappaB activation were sensitive to pertussis but not cholera toxin, suggesting that the B2 BK receptors transducing the response were coupled to Galphai or Galphao heterotrimeric G proteins. Tumor necrosis factor alpha (TNFalpha) also stimulated increased PI 3-kinase activity, however TNFalpha-stimulated NF-kappaB activation was not affected by the PI 3-kinase inhibitors or the p85 dominant negative mutant. These findings provide evidence that BK-induced NF-kappaB activation utilizes a signaling pathway that requires activity of both RhoA and PI 3-kinase and is distinct from the signaling pathway utilized by TNFalpha. Furthermore, we show that the p85 regulatory subunit is required for activation of PI 3-kinase activity by this G protein-coupled receptor.

KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development.

Cell adhesion molecules (CAMs) are important mediators of cell-cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell-cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.

Phosphatidylinositol 3-kinase is a negative regulator of cellular differentiation.

Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor-induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.

D-myo-Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-kinase signaling pathways.

Several inositol-containing compounds play key roles in receptor-mediated cell signaling events. Here, we describe a function for a specific inositol polyphosphate, D-myo-inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P4], that is produced acutely in response to a receptor-independent process. Thus, infection of intestinal epithelial cells with the enteric pathogen Salmonella, but not with other invasive bacteria, induced a multifold increase in Ins(1,4,5,6)P4 levels. To define a specific function of Ins(1,4,5,6)P4, a membrane-permeant, hydrolyzable ester was used to deliver it to the intracellular compartment, where it antagonized epidermal growth factor (EGF)-induced inhibition of calcium-mediated chloride (Cl-) secretion (CaMCS) in intestinal epithelia. This EGF function is likely mediated through a phosphoinositide 3-kinase (PtdIns3K)-dependent mechanism because the EGF effects are abolished by wortmannin, and three different membrane-permeant esters of the PtdIns3K product phosphatidylinositol 3,4,5-trisphosphate mimicked the EGF effect on CaMCS. We further demonstrate that Ins(1,4,5,6)P4 antagonized EGF signaling downstream of PtdIns3K because Ins(1,4,5, 6)P4 interfered with the PtdInsP3 effect on CaMCS without affecting PtdIns3K activity. Thus, elevation of Ins(1,4,5,6)P4 in Salmonella-infected epithelia may promote Cl- flux by antagonizing EGF inhibition mediated through PtdIns3K and PtdInsP3.

A tyrosine kinase signaling pathway accounts for the majority of phosphatidylinositol 3,4,5-trisphosphate formation in chemoattractant-stimulated human neutrophils.

The signaling pathway leading from G protein-coupled chemoattractant receptors to the generation of oxidants by NADPH oxidase in human neutrophils requires the formation of the lipid mediator phosphatidylinositol 3,4,5-trisphosphate (PIP3). Two mechanisms through which PIP3 can be generated have been described in human leukocytes. One pathway involves the coupling of the src-related tyrosine kinase Lyn to the "classical" p85/p110 form of phosphatidylinositol 3-kinase. The second paradigm utilizes a novel form of phosphatidylinositol 3-kinase whose activity is directly regulated by G protein betagamma subunits. In this paper, we show that formation of PIP3 in chemoattractant-stimulated neutrophils is substantially attenuated by inhibitors that specifically block tyrosine kinase activity. These data suggest that the Lyn activation pathway plays a major role in the formation of this important lipid messenger during chemoattractant stimulation of human neutrophils.

G protein-coupled chemoattractant receptors regulate Lyn tyrosine kinase.Shc adapter protein signaling complexes.

Receptors for chemoattractants that direct the migration of phagocytic leukocytes to sites of injury/infection also modulate many other leukocyte functions that are critical to the inflammatory response. These chemoattractant receptors, members of the G protein-coupled heptahelical receptor family, have been classically linked to cell activation via phospholipase C, calcium, and protein kinase C. We show here that activation of the N-formyl peptide chemoattractant receptor stimulates an additional protein kinase C-independent pathway through the Src-related tyrosine kinase, Lyn, in human neutrophils. We demonstrate that activation of Lyn is associated with binding to the Shc adapter protein, which becomes phosphorylated on tyrosine residues. This interaction appears to be mediated via the Shc SH2 domain. Complexes of phosphorylated Lyn and Shc with phosphatidylinositol 3-kinase are rapidly formed in stimulated neutrophils, correlating with phosphatidylinositol 3,4,5-trisphosphate [corrected] formation and cell activation. This signaling pathway involving a Src-related kinase and the Shc adapter protein provides a potential mechanism linking chemoattractant receptors to downstream events involving Rac activation and NADPH oxidase. Regulation of Shc by G protein-coupled receptors may also allow these receptors to modulate the activity of the Ras/mitogen-activated protein kinase cascade.

Failure of senescent human fibroblasts to express the insulin-like growth factor-1 gene.

Senescent human diploid fibroblasts express several growth-regulated genes but fail to express others. In this paper we show, by a very sensitive technique (reverse transcriptase-polymerase chain reaction), that senescent cells fail to express insulin-like growth factor-1 (IGF-1) mRNA, which is expressed in moderate amounts by young cells. Human fibroblasts immortalized by transfection with a temperature-sensitive SV40 T antigen gene regain the ability to express IGF-1 mRNA, but only at the permissive temperature of 34 degrees C. Under these conditions, the immortalized human fibroblasts grow even in 1% serum. At the restrictive temperature of 39 degrees C, the temperature-sensitive T antigen is nonfunctional, IGF-1 RNA is not detectable, and the cells fail to grow even in 10% serum. The failure to express IGF-1 mRNA in postsenescent cells can be ascribed, at least in part, to a transcriptional mechanism. Despite the correlation among immortalization by SV40 T antigen, expression of IGF-1, and growth, it seems unlikely that the failure to express IGF-1 is the sole cause of cellular senescence; other requirements must be postulated.

IGF-1 receptor levels and the proliferation of young and senescent human fibroblasts.

We have investigated the role of the IGF-1 receptor in the proliferation of young and senescent human diploid fibroblasts. Using WI-38 cells, we have established the following: 1) both young and senescent cells have IGF-1 receptors, which can be autophosphorylated by IGF-1, the intensity of the autophosphorylation being roughly the same in both types of cells; 2) the levels of IGF-1 receptor mRNA are also similar in young and senescent cells; 3) both young and senescent cells have an absolute requirement for the IGF-1 receptor in order to be stimulated by either serum or SV40, respectively; 4) despite these similarities, young cells respond to IGF-1 (in combination with other growth factors) with DNA synthesis and mitosis, and senescent cells do not. We conclude that, although the IGF-1 receptor is still needed by senescent cells for a growth response to SV40, it is not, by itself, the determinant of senescence, at least in WI-38 cells.

Effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation of young and senescent WI-38 human diploid fibroblasts.

The effects of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on the proliferation, protein kinase C activity (PKC), and c-fos gene expression were examined in cultures of young and senescent (90-95% lifespan completed) WI-38 human diploid fibroblasts. We observed that, following stimulation with medium containing 10% fetal bovine serum (FBS), the translocation of PKC from the cytosol to the particulate compartment was less efficient in senescent WI-38 cells than in young cells. However, when PMA was added to the medium, the intracellular distribution of PKC activity in old cells became nearly identical to that observed in young cells. The inducibility of c-fos mRNA by serum addition, which is a protein kinase C-dependent event, was significantly amplified in the presence of PMA. Moreover, the duration of peak c-fos expression, after stimulation by FBS and PMA, increased in senescent cells as compared to young cells. Our results reveal that the normal signal transduction pathway is altered in senescent, slowly proliferating human fibroblasts and that it can be partially restored in the presence of the tumor promoter PMA.

CSF-1 deficiency in the op/op mouse has differential effects on macrophage populations and differentiation stages.

Osteopetrosis and the absence of colony-stimulating factor 1 (CSF-1) in op/op mice are associated with decreased cellularity of the bone marrow (to one tenth of the normal), a very significant reduction in the number of cells recovered from peritoneal, pleural, and alveolar lavages, moderate leukopenia, and a slight decrease in the number of cells per spleen and thymus. Furthermore, op/op mice possess deficiencies in the number of macrophages in various organs. These cells are apparently absent in the bone marrow, severely reduced (5%-15% of the normal number) in peritoneal and pleural cavities and in the lungs. In addition, a marked decrease in the frequency and total number of circulating monocytes is present (5% of the normal). The deficiency of macrophages is less severe in the liver, spleen, and thymus of op/op mice (approximately 30% of those seen in normal). There is a concomitant redistribution of macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) in op/op mice from the marrow to the spleen and liver, associated with an increased sensitivity to interleukin 3 (IL-3). Their total number is decreased at least threefold compared to control mice. Moreover, op/op mice have at least a fivefold reduction in the total number of day-11 spleen colony-forming units (CFU-S) associated with their redistribution to the spleen and liver. These data suggest that the macrophage system in op/op mice is reduced at all levels tested, that is, at the level of mature macrophages, the level of progenitors, and the level of stem cells, whereas the redistribution of progenitor and stem cells could be viewed as a secondary consequence of osteopetrosis. Furthermore, these data suggest that macrophage dependency in vivo on CSF-1 is limited and different in various organs. Particularly in the liver, spleen, and thymus, other growth factors may significantly compensate for CSF-1 deficiency. Based on the relative decrease in the number of CFU-GM in the op/op mice, it appears that the population size of these progenitors is less dependent on CSF-1 than the hematopoietic stem cell population size as evidenced by the day-11 CFU-S assay. The day-11 CFU-S population is severely reduced in op/op mice, suggesting a physiological involvement of CSF-1 in expanding its size. These data provide evidence that CSF-1, besides acting on the final and intermediate stages of macrophage maturation, may also play a role in early stages of hematopoiesis.

Adherent cell growth from murine bone marrow in liquid cultures. Inbred strain variability and effects of mutations affecting hemopoiesis.

The marrow cells of mice from seven hematologically normal strains (AKR/J, AKR/Cum, CBA/J, C3H/HeJ, C57B1/6J, DBA/2J, and RF/J) and of mice with five mutations in loci affecting hemopoiesis (W, Sl, nu, xid, NZB) were tested. The following parameters of adherent cell growth in 14 day liquid culture were analyzed: number and diameter of macroscopic adherent colonies, cellular composition with particular reference to the number of macrophage-like cells attached to a single fibroblast-like cell on peripheries of adherent colonies and the number of macrophage-like cells per eyefield of intercolony spaces. No qualitative differences were observed between tested genotypes in the cellular composition of adherent layers. These layers were uniformly made of colonies of fibroblast-like cells that were overgrown by macrophage-like cells. Macrophage-like cells grew also in intercolony spaces but this never occurred to fibroblast-like cells. At least using light microscopy and Wright's staining no morphological differences could be observed between either fibroblast-like cells or macrophage-like cells derived from marrow of various murine strains. This is of particular significance for Sl/Sld mice that suffer from the functional defect of hemopoietic microenvironment. On the other hand, large strain variability was observed in the number (5-16/10(6) marrow cells, 10.6 on the average) and diameter (1.9-3.7 mm, 2.9 mm on the average) of macroscopic adherent colonies. Among mutant genotypes, only NZB mice and animals with xid mutation formed significantly increased numbers of macroscopic colonies, while values for other mutant genotypes, Sl/Sld included, did not differ from normal littermate controls.

The effects of incubation of marrow in tissue fragments in vitro on the growth of adherent cells from this marrow.

Femoral marrow was either cultured as a single cell suspension immediately following collection from the donor mouse or following 4 day incubation in vitro of the whole marrow shaft. Several parameters of growth of adherent, i.e. composed of fibroblastoid cells and macrophages, colonies were determined following 14 day culture in Dulbecco medium at 37 degrees C. These included: number and diameter of macroscopic colonies, number of macrophages per fibroblastoid cell inside the colonies and per eyefield in intercolony spaces, number of cells in supernatant from the culture. The 4 day incubation of marrow fragments in vitro (Dulbecco medium, 37 degrees C) doubled the number of adherent colonies grown from this marrow and, moreover, the colonies formed were increased in size. Other parameters of cell growth in these cultures were unchanged. These data suggest that under conditions of in vitro incubation of marrow shaft (close cell-to-cell contact) marrow fibroblastoid colony forming units (MF-CFU) are stimulated to self-renewal.