Nerve growth factor induces increased expression of a laminin-binding integrin in rat pheochromocytoma PC12 cells

Rat pheochromocytoma PC12 cells exposed to nerve growth factor differentiate as sympathetic neurons and extend neurites on laminin and to a much lesser extent on fibronectin. Analysis of laminin fragments indicated that neurite outgrowth occurs mainly on fragment P1, corresponding to the center of the cross, and only poorly on fragment E8, a long arm structure that is active with other neuronal cells. Integrin antibodies prevented adhesion and neurite sprouting of these cells on laminin, fragment P1, and fibronectin. By affinity chromatography we isolated an integrin-type receptor for laminin consisting of two subunits with molecular massess of 180 and 135 kDa. The latter is recognized by an antiserum to integrin beta 1 subunit. The bound laminin receptor could be displaced by EDTA, but not by Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg peptides. Affinity chromatography on laminin fragments showed that the 180/135 kDa receptor binds to P1. The expression of the 180-kDa alpha subunit of the laminin receptor at the cell surface was increased 10-fold after NGF treatment. The effect of NGF is specific since the amount of a 150-kDa fibronectin-binding integrin alpha subunit remained unchanged. Moreover, the increased expression of the 180/135 kDa receptor at the cell surface corresponded to a selective increase in cell adhesion to laminin and to fragment P1. The 180/135-kDa complex is thus an integrin-type receptor for laminin whose expression and binding specificity correlates with the capacity of NGF-induced PC12 cells to extend neurites on laminin.     

Nerve growth factor induces 5-HT3 recognition sites in rat pheochromocytoma (PC12) cells

In rat pheochromocytoma (PC12) cells, nerve growth factor (7S NGF) induced the expression of recognition sites that bind the specific 5-HT3 antagonist (S-) [3H]zacopride. Culturing PC12 cells for 8-12 days in the presence of 50 ng/ml NGF increased the density (Bmax) of (S-) [3H]zacopride binding sites in cell membranes (0-100,000 x g fraction) from 0 to 105 fmoles/mg protein. This binding exhibited high affinity for (S-) [3H]zacopride (Kd = 0.8 nM), was specific (greater than 95%), and was inhibited by 5-HT3 compounds with a rank of potency (quipazine greater than ICS 205-930 greater than GR38032F greater than BRL24924 approximately MDL 72222 greater than phenylbiguanide greater than or equal to serotonin greater than 2-methyl-serotonin greater than metoclopramide) which was distinct from neuroblastoma cells. Thus, NGF-differentiated PC12 cells possess a 5-HT3 receptor and should be useful to investigate its regulation and biochemical mechanism of action.     

Nerve growth factor stimulates phosphorylation of phospholipase C-gamma in PC12 cells.

PC12 cells contain at least three immunologically distinct phospholipase C (PLC) isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of PC12 cells with nerve growth factor (NGF) leads to an increase in the phosphorylation of PLC-gamma, but not of PLC-beta or PLC-delta. This increase can be seen in as little as 1 minute. The increased phosphorylation occurs on both serine and tyrosine residues, with the major increase being in the former. This result suggests the possibility that the NGF-dependent increase in phosphoinositide hydrolysis in PC12 cells is due to selective phosphorylation of PLC-gamma by serine and tyrosine protein kinases associated with the NGF receptor.     

Nerve growth factor enhances expression of neuron-glia cell adhesion molecule in PC12 cells

The neuron-glia cell adhesion molecule (Ng-CAM) has been identified in mammalian brain tissue and PC12 pheochromocytoma cells as Mr 200,000 and Mr 230,000 species, respectively. When PC12 cells were treated with nerve growth factor (NGF), the amount of Ng-CAM at the cell surface was increased approximately threefold, whereas the amount of the neural cell adhesion molecule (N-CAM) remained unchanged. An NGF-inducible large external glycoprotein (NILE) has been previously identified by its enhanced expression in NGF-treated PC12 cells. Ng-CAM and NILE are similar in molecular weight, expression during development, and responsiveness to NGF in PC12 cells, suggesting that the two molecules are related. In addition, antibodies to Ng-CAM and NILE cross-reacted and the molecules had similar peptide maps after limited proteolysis. Moreover, antibodies to Ng-CAM inhibited fasciculation of neurites, a functional property shared with NILE. The results show that cell adhesion molecules can respond selectively to growth factors and suggest that NILE is, in fact, mammalian Ng-CAM.     

Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.     

Nerve growth factor regulates the abundance and distribution of K+ channels in PC12 cells

We examined the effect of nerve growth factor (NGF) treatment on expression of a neuronal delayed rectifler K+ channel subtype, Kv2.1 (drk1), in PC12 cells. Anti-Kv2.1 antibodies recognized a single polypeptide population of M(r) = 132 kD in PC12 cell membranes, distinct from the more heterogeneous population found in adult rat brain. In response to NGF treatment, levels of Kv2.1 polypeptide in PC12 membranes increased fourfold. This increase in polypeptide levels could be seen within 12 h, and elevated levels were maintained for at least 6 d of continuous NGF treatment. RNase protection assays indicate that this increase in Kv2.1 protein occurs without an increase in steady state levels of Kv2.1 mRNA following NGF treatment. Immunofluorescent localization of the Kv2.1 polypeptide revealed plasma membrane-associated staining of cell bodies in both untreated and NGF- treated PC12 cells. In undifferentiated cells, intense staining is seen at sites of cell-cell and cell-substratum contact. In differentiated cells the most intense Kv2.1 staining is observed in neuritic growth cones. These studies show that in PC12 cells both the abundance and distribution of the Kv2.1 k+ channel are regulated by NGF, and suggest that PC12 cells provide a model for the selective expression of Kv2.1 in neuritic endings.     

Nerve growth factor stimulates the tyrosine phosphorylation of MAP2 kinase in PC12 cells.

NGF treatment of PC12 cells results in the rapid activation of MAP2 kinase. We report here that the induction of enzyme activity was correlated with the phosphorylation of MAP2 kinase, detected by metabolic labeling of the enzyme and with anti-phosphotyrosine antibodies. NGF stimulated the phosphorylation of MAP2 kinase on tyrosine, as well as serine and threonine residues. Western blot analysis using a polyclonal anti-phosphotyrosine antibody demonstrated that the tyrosine phosphorylation of MAP2 kinase was maximal within 2 min following NGF exposure and preceded the induction of MAP2 kinase activity. The NGF-stimulated tyrosine phosphorylation of an identified substrate provides direct evidence for the participation of a tyrosine kinase in the mechanism of action of NGF.     

Requirement for specific protein kinase activities during the rapid redistribution of F-actin that precedes the outgrowth of neurites in PC12D cells.

Rapid changes in morphology of PC12D cells, a subline of PC12 cells, in response to various agents were studied in relation to the subsequent outgrowth of neurites. A few minutes after addition of NGF or of dbcAMP, staining of F-actin with rhodamine phalloidin revealed the formation of ruffles around the periphery of cells. Simultaneous relocalization of F-actin to the area of ruffles occurred in response to NGF. A moderate relocalization of F-actin occurred in dbcAMP-treated cells. Other neurite-promoting agents on PC12D cells, such as bFGF, EGF and PMA, also caused ruffling and an identical redistribution of F-actin. The actin bundles then condensed into several dot-like aggregates that subsequently became the growth cones of neurites. When an inhibitor of protein kinase, K-252a, was added, only the NGF-induced morphological change was selectively decreased. By contrast, an inhibitor of protein kinase A, H-89, selectively blocked the dbcAMP-induced change. These are analogous to the effects of those inhibitors on the outgrowth of neurites. These observations indicate that the formation of ruffles with the redistribution of F-actin might be one of the earliest steps in the neurite outgrowth and that the morphological changes might be triggered by the activation of specific protein kinases. Neither cytochalasin B nor colchicine prevented the series of morphological changes. However, processes formed in the presence of cytochalasin B had no filopodium and protrusions formed in the presence of colchicine were shaped like large filopodia. It appears that microtubules and microfilaments may not be absolutely required for the initiation of the rapid morphological changes, but that complete neurites might be formed with contribution by microtubules and by microfilaments.