Protein tyrosine phosphatase activation during nerve growth factor-induced neuronal differentiation of PC12 cells.

We have studied the role of protein tyrosine phosphatases (PTPases) during neuronal differentiation of PC12 cells. Nerve growth factor (NGF), a well-characterized differentiating agent for these cells, led to a decrease in DNA synthesis within 24 h. This was accompanied by a 2- to 3-fold increase in the activity of PTPases, measured as the dephosphorylation of polyacidic or polybasic substrates phosphorylated on tyrosine. PTPase activation was independent of cell density and proportional to NGF concentration, with a half-maximal effect occurring at 0.35 nM. High-performance liquid chromatography size exclusion chromatography revealed that PTPases with molecular masses of 550, 300, and 60 kilodaltons were activated in response to NGF. Additional studies showed that the presence of NGF made PC12 cells refractory to the mitogenic effect of epidermal growth factor. Our data indicate that NGF-induced neuronal differentiation and growth arrest in PC12 cells are associated with activation of several PTPases. We speculate that PTPase activation in response to NGF may inhibit the mitogenic actions of other growth factors.     

Activation of phosphotyrosine phosphatase activity is associated with decreased differentiation in adult bovine lens

The postnatal vertebrate eye lens provides an opportunity to study possible involvement of reversible protein phosphorylation in the differentiation process of epithelial cells. Epithelial cells at the lens equator, indeed, differentiate continuously into fiber cells throughout life but this capacity progressively decreases with age. Here we describe the characterization of a phosphotyrosine-protein phosphatase(s) (PTPase(s)) in the equatorial epithelium of bovine lens which exhibits a high level of specific activity. PTPase(s) is detected in cellular detergent extracts using phospholabeled synthetic peptides, p-nitrophenyl phosphate, and lens epithelial membranes as substrates. We show that activity of this PTPase(s) is increased in the equatorial epithelium as the age is increased. We also show that this enzyme(s) exerts its dephosphorylating activity predominantly on a calpactin-like protein associated with lens epithelial membranes. Dephosphorylation of this protein is only obtained when membranes are subjected to extracts in the presence of fibroblast growth factor (FGF). It is suggested that an FGF-activated PTPase(s) might conceivably counteract effects of differentiation stimulatory factors for limiting differentiation of lens throughout life. © 1995 Wiley-Liss, Inc.     

Protein-tyrosine phosphatases and the regulation of insulin action.

Protein-tyrosine phosphatases (PTPases) play an important role in the regulation of insulin action by dephosphorylating the active (autophosphorylated) form of the insulin receptor and attenuating its tyrosine kinase activity. PTPases can also modulate post-receptor signalling by catalyzing the dephosphorylation of cellular substrates of the insulin receptor kinase. Dramatic advances have recently been made in our understanding of PTPases as an extensive family of transmembrane and intracellular proteins that are involved in a number of pathways of cellular signal transduction. Identification of the PTPase(s) which act on various components of the insulin action cascade will not only enhance our understanding of insulin signalling but will also clarify the potential involvement of PTPases in the pathophysiology of insulin-resistant disease states. This brief review provides a summary of reversible tyrosine phosphorylation events in insulin action and available data on candidate PTPases in liver and skeletal muscle that may be involved in the regulation of insulin action.     

Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.     

The stability of the lens-specific Maf protein is regulated by fibroblast growth factor (FGF)/ERK signaling in lens fiber differentiation

Fibroblast growth factor (FGF) signaling is necessary for both proliferation and differentiation of lens cells. However, the molecular mechanisms by which FGFs exert their effects on the lens remain poorly understood. In this study, we show that FGF-2 repressed the expression of lens-specific genes at the proliferative phase in primary cultured lens cells. Using transfected cells, we also found that the activity of L-Maf, a lens differentiation factor, is repressed by FGF/ERK signaling. L-Maf is shown to be phosphorylated by ERK, and introduction of mutations into the ERK target sites on L-Maf promotes its stabilization. The stable L-Maf mutant protein promotes the differentiation of lens cells from neural retina cells. Taken together, these results indicate that FGF/ERK signaling negatively regulates the function of L-Maf in proliferative lens cells and that stabilization of the L-Maf protein is important for lens fiber differentiation.     

Factors modulating mouse lens epithelial cell morphology with differentiation and development of a lentoid structure in vitro

The morphological and cellular changes that occur with differentiation and development of a lentoid structure from cultured mouse lens epithelial cells have been found to be dependent on the presence of lens capsule in association with the cells. The development of the 'lentoid body' is a multiphase process involving cell replication, synthesis of mucosubstances and a basement collagen membrane, cell aggregation and differentiation. Stage-specific synthesis of lens proteins confirms that the genes regulating normal differentiation in vivo are operating in the in vitro system. The hydrated collagen gel studies described in this report demonstrate that the cuboidal morphology and apical-basal polarity of the lens epithelial cells are dependent on their relationship with the lens capsule. Following a replicative phase the cells assume a mesenchyme-like morphology and migrate into the gel. Trypsinized cells freed from the lens capsule replicate but form colonies on the surface of the gel. The implications of these results are discussed with respect to previous observations made on normal lens development and the abnormalities associated with the congenital cataractous embryonic lens.     

Receptor protein tyrosine phosphatase alpha activates pp60c-src and is involved in neuronal differentiation.

Here we report that protein tyrosine phosphatases (PTPases), like their enzymatic counterpart the protein tyrosine kinases, can play an important role in cell differentiation. Expression of the transmembrane PTPase receptor protein tyrosine phosphatase alpha (RPTP alpha) is transiently enhanced during neuronal differentiation of embryonal carcinoma (EC) and neuroblastoma cells. Retinoic acid induces wild type P19 cells to differentiate into endoderm- and mesoderm-like cells. By contrast, retinoic acid treatment leads to neuronal differentiation of P19 cells, ectopically expressing functional RPTP alpha, as illustrated by their ability to generate action potentials. Endogenous pp60c-src kinase activity is enhanced in the RPTP alpha-transfected cells, which may be due to direct dephosphorylation of the regulatory Tyr residue at position 527 in pp60c-src by RPTP alpha. Our results demonstrate that RPTP alpha is involved in neuronal differentiation and imply a role for pp60c-src in the differentiation process.     

Ras signaling is essential for lens cell proliferation and lens growth during development

The vertebrate ocular lens is a simple and continuously growing tissue. Growth factor-mediated receptor tyrosine kinases (RTKs) are believed to be required for lens cell proliferation, differentiation and survival. The signaling pathways downstream of the RTKs remain to be elucidated. Here, we demonstrate the important role of Ras in lens development by expressing a dominant-negative form of Ras (dn-Ras) in the lens of transgenic mice. We show that lens in the transgenic mice was smaller and lens growth was severely inhibited as compared to the wild-type lens. However, the lens shape, polarity and transparency appeared normal in the transgenic mice. Further analysis showed that cell proliferation is inhibited in the dn-Ras lens. For example, the percentage of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in epithelial layer was about 2- to 3-fold lower in the transgenic lens than in the wild-type lens, implying that Ras activity is required for normal cell proliferation during lens development. We also found a small number of apoptotic cells in both epithelial and fiber compartment of the transgenic lens, suggesting that Ras also plays a role in cell survival. Interestingly, although there was a delay in primary fiber cell differentiation, secondary fiber cell differentiation was not significantly affected in the transgenic mice. For example, the expression of beta- and gamma-crystallins, the marker proteins for fiber differentiation, was not changed in the transgenic mice. Biochemical analysis indicated that ERK activity, but not Akt activity, was significantly reduced in the dn-Ras transgenic lenses. Overall, our data imply that the RTK-Ras-ERK signaling pathway is essential for cell proliferation and, to a lesser extent, for cell survival, but not for crystallin gene expression during fiber differentiation. Thus, some of the fiber differentiation processes are likely mediated by RTK-dependent but Ras-independent pathways.     

PRL-1 PTPase expression is developmentally regulated with tissue-specific patterns in epithelial tissues

The mechanisms controlling tyrosine phosphorylation of cellular proteins are important in the regulation of many cellular processes, including development and differentiation. Protein tyrosine phosphatases (PTPases) may be as important as protein tyrosine kinases (PTKs) in these processes. PRL-1 is a distinct PTPase originally identified as an immediate-early gene in liver regeneration whose expression is associated with growth in some tissues but with differentiation in others. We now demonstrate that the PRL-1 protein is expressed during development in a number of digestive epithelial tissues. It is expressed at variable time points in the developing intestine, but its expression is limited to the developing villus enterocytes. In the gastric epithelium, PRL-1 expression in the adult is restricted to zymogen cells. PRL-1 is also expressed in the developing liver and esophagus and in the epithelia of the kidney and lung. In each of these contexts, the expression of PRL-1 is associated with terminal differentiation, suggesting that it may play a role in this important developmental process.     

The canonical intrinsic mitochondrial death pathway has a non-apoptotic role in signaling lens cell differentiation

The mitochondrial cell death pathway is known for its role in signaling apoptosis. Here, we describe a novel function for the mitochondrial cell death pathway in signaling initiation of differentiation in the developing lens. Most remarkably, we induced lens cell differentiation by short-term exposure of lens epithelial cells to the apoptogen staurosporine. Activation of apoptosis-related pathways induced lens epithelial cells to express differentiation-specific markers and to undergo morphogenetic changes that led to formation of the lens-like structures known as lentoids. The fact that multiple stages of differentiation are expressed at a single stage of development in the embryonic lens made it possible to precisely determine the timing of expression of proteins associated with the apoptotic pathway. We discovered that there was high expression in the lens equatorial epithelium (the region of the lens in which differentiation is initiated) of pro-apoptotic molecules such as Bax and Bcl-x(S) and release of cytochrome c from mitochondria. Furthermore, we found significant caspase-3-like activity in the equatorial epithelium, yet this activity was far lower than that associated with lens cell apoptosis. These apoptotic pathways are likely regulated by the concurrent expression of prosurvival molecules, including Bcl-2 and Bcl-x(L); phosphorylation of Bad; and high expression of inhibitor of apoptosis proteins chicken IAP1, IAP3, and survivin. This finding suggests that prosurvival pathways allow pro-apoptotic molecules to function as molecular switches in the differentiation process without tipping the balance toward apoptosis. We call this process apoptosis-related Bcl-2- and caspase-dependent (ABC) differentiation.     

Transient Association of the Phosphotyrosine Phosphatase SHP-2 with TrkA Is Induced by Nerve Growth Factor

Abstract: Nerve growth factor (NGF) treatment of rat PC12 pheochromocytoma cells results in an increase in the tyrosine phosphorylation of the NGF receptor, TrkA, leading to differentiation to a neuronal phenotype. Dephosphorylation by protein tyrosine phosphatases (PTPases) is thought to play an important role in regulating this signaling pathway. To identify PTPases that are recruited to the activated TrkA receptor, we used an ingel PTPase assay to examine the presence of PTPases in TrkA immunoprecipitates. The Src homology 2 domain containing PTPase SHP-2 was found to associate transiently with TrkA following receptor activation, reaching a peak after 1 min of NGF treatment and then decreasing rapidly. The association of SHP-2 with TrkA was accompanied by the tyrosine phosphorylation of SHP-2 and an association of SHP-2 with multiple tyrosine-phosphorylated proteins. In addition, the PTPase activity in SHP-2 immunoprecipitates increased greater than twofold after 1 min of NGF treatment. This is the first demonstration that the association of SHP-2 with TrkA is induced by NGF and that this association leads to SHP-2 activation and tyrosine phosphorylation. We conclude that SHP-2 plays a significant role in early biochemical events in TrkA-mediated signal transduction.     

Regulation of the insulin signalling pathway by cellular protein-tyrosine phosphatases

Protein-tyrosine phosphatases (PTPases) have been implicated in the physiological regulation of the insulin signalling pathway. In cellular and molecular studies, the transmembrane, receptor-type PTPase LAR and the intracellular, non-receptor enzyme PTP1B have been shown to have a direct impact on insulin action in intact cell models. Since insulin signalling can be enhanced by reducing the abundance or activity of specific PTPases, pharmaceutical agents directed at blocking the interaction between individual PTPases and the insulin receptor may have potential clinical relevance to the treatment of insulin-resistant states such as obesity and Type II diabetes mellitus.     

Inhibition of lens fiber cell morphogenesis by expression of a mutant SV40 large T antigen that binds CREB-binding protein/p300 but not pRb

Simian virus (SV) 40 large T antigen can both induce tumors and inhibit cellular differentiation. It is not clear whether these cellular changes are synonymous, sequential, or distinct responses to the protein. T antigen is known to bind to p53, to the retinoblastoma (Rb) family of tumor suppressor proteins, and to other cellular proteins such as p300 family members. To test whether SV40 large T antigen inhibits cellular differentiation in vivo in the absence of cell cycle induction, we generated transgenic mice that express in the lens a mutant version of the early region of SV40. This mutant, which we term E107KDelta, has a deletion that eliminates synthesis of small t antigen and a point mutation (E107K) that results in loss of the ability to bind to Rb family members. At embryonic day 15.5 (E15.5), the transgenic lenses show dramatic defects in lens fiber cell differentiation. The fiber cells become post-mitotic, but do not elongate properly. The cells show a dramatic reduction in expression of their beta- and gamma-crystallins. Because CBP and p300 are co-activators for crystallin gene expression, we assayed for interactions between E107KDelta and CBP/p300. Our studies demonstrate that cellular differentiation can be inhibited by SV40 large T antigen in the absence of pRb inactivation, and that interaction of large T antigen with CBP/p300 may be enhanced by a mutation that eliminates the binding to pRb.     

DIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN TISSUE CULTURE (III) FUNCTIONS IN VITRO OF A TRANSFORMED RAT LENS EPITHELIAL CELL LINE

Epithelial cells isolated from one-day-old rat lens were neoplastically transformed using Rous Sarcoma Virus (Schmidt-Ruppin-D strain) and have been in continuous culture for over 30 months. This cell line displays some of the characteristics of lens epithelial cells, but can be shown to represent a permanent cell line in that it has been transferred more than 50 times and is continuing to grow, while primary lens cell cultures have never survived beyond 8 weeks. The infecting viral genome can be rescued from the cell line upon fusion with uninfected chick embryo cells. It is anticipated that this newly established cell line, designated RLE-R, will be useful in studies on cellular differentiation.     

Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini.

Eukaryotic cells respond to various stimuli by an increase or decrease in levels of phosphoproteins. Phosphotyrosine levels on eukaryotic cellular proteins are tightly regulated by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases, EC 3.1.3.48). Studies on permeabilized mast cells suggest that the enabling reaction for exocytosis might involve protein dephosphorylation. In the present studies, a recombinant form of rat brain PTPase (rrbPTP-1) has been used to examine the potential role of PTPases in Ca(2+)-dependent amylase secretion from permeabilized rat pancreatic acini. Additionally, the concentrations and subcellular distributions of endogenous PTPase activity in rat pancreas were determined. The results from these experiments indicate that addition of exogenous PTPase stimulated Ca(2+)-dependent amylase secretion from pancreatic acinar cells and that endogenous PTPase activity was associated with the postgranule supernatant, zymogen granules, and in particular zymogen granule membranes. Our data suggest that protein tyrosine dephosphorylation is potentially involved in regulated secretion at a site(s) distal to receptor-mediated elevation of intracellular second messengers.     

Decreased turnover of phosphatidylinositol accompanies in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers

The in vivo differentiation of embryonic chicken lens epithelial cells into lens fibers is accompanied by a marked decrease in the rate of degradation of phosphatidylinositol. The present experiments were undertaken to determine whether a similar change in phosphatidylinositol metabolism occurs during in vitro lens fiber formation in cultured explants of embryonic chicken lens epithelia. Lens epithelial cells in the explants differentiate into lens fibers following the addition of fetal calf serum, insulin or chicken vitreous humor to the culture medium. The results show that phosphatidylinositol is degraded with a half-life of 3-6 h in cultured lens epithelia that are not stimulated to differentiate. In contrast, no degradation occurs for at least 6 h in lens epithelia stimulated to form lens fibers. The stabilization of phosphatidylinositol is apparent within 4 h after the onset of fiber cell formation, and thus represents an early event in differentiation. The rapid degradation of phosphatidylinositol in lens epithelia is accompanied by comparably rapid synthesis. During this metabolic turnover only the phosphorylinositol portion of the molecule is renewed, as expected if hydrolysis occurs by the action of a phospholipase C, such as phosphatidylinositol phosphodiesterase. Thus, these data suggest that agents which produce in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers lead to a reduction in either the amount or the activity of phospholipase C.     

Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade

The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key post-receptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H(2)O(2) in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H(2)O(2) as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and high M(r) insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases.