Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth.

We have studied the role of vinculin in regulating integrin-dependent neurite outgrowth in PC12 cells, a neuronal cell line. Vinculin is a cytoskeletal protein believed to mediate interactions between integrins and the actin cytoskeleton. In differentiated PC12 cells, the cell body, the neurite, and the growth cone contain vinculin. Within the growth cone, both the proximal region of "consolidation" and the more distal region consisting of lamellipodia and filopodia contain vinculin. To study the role of vinculin in neurite outgrowth, we generated vinculin-deficient isolates of PC12 cell lines by transfection with vectors expressing antisense vinculin RNA. In some of these cell lines, vinculin levels were reduced to 18-23% of normal levels. In the vinculin-deficient cell lines, neurite outgrowth on laminin was significantly reduced. In time-lapse analysis, growth cones advanced much more slowly than normal. Further analysis indicated that this deficit could be explained in large part by changes in the behaviors of filopodia and lamellipodia. Filopodia were formed in normal numbers, extended at normal rates, and extended to approximately normal lengths, but were much less stable in the vinculin deficient compared to control PC12 cells. Similarly, lamellipodia formed and grew nearly normally, but were dramatically less stable in the vinculin-deficient cells. This can account for the reduction in rate of growth cone advance. These results indicate that interactions between integrins and the actin-based cytoskeleton are necessary for stability of both filopodia and lamellipodia.

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Goosecoid suppresses cell growth and enhances neuronal differentiation of PC12 cells

Journal of Cell Science ◽

10.1242/jcs.113.15.2705 ◽

2000 ◽

Vol 113 (15) ◽

pp. 2705-2713

Author(s):

K. Sawada ◽

Y. Konishi ◽

M. Tominaga ◽

Y. Watanabe ◽

J. Hirano ◽

...

Keyword(s):

Transcription Factor ◽

Neurite Outgrowth ◽

Neuronal Differentiation ◽

Pc12 Cells ◽

Mammalian Cells ◽

Homeobox Gene ◽

S Phase ◽

Bhlh Transcription Factor ◽

Spemann Organizer

In all vertebrate species, the homeobox gene goosecoid serves as a marker of the Spemann organizer tissue. One function of the organizer is the induction of neural tissue. To investigate the role of goosecoid in neuronal differentiation of mammalian cells, we have introduced goosecoid into PC12 cells. Expression of goosecoid resulted in reduced cell proliferation and enhanced neurite outgrowth in response to NGF. Expression of goosecoid led to a decrease in the percentage of S-phase cells and to upregulation of the expression of the neuron-specific markers MAP-1b and neurofilament-L. Analysis of goosecoid mutants revealed that these effects were independent of either DNA binding or homodimerization of Goosecoid. Coexpression of the N-terminal portion of the ets transcription factor PU.1, a protein that can bind to Goosecoid, repressed neurite outgrowth and rescued the proliferation of PC12 cultures. In contrast, expression of the bHLH transcription factor HES-1 repressed goosecoid-mediated neurite outgrowth without changing the proportion of S-phase cells. These results suggest that goosecoid is involved in neuronal differentiation in two ways, by slowing the cell cycle and stimulating neurite outgrowth, and that these two events are separately regulated.

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Characterization of a PC12 cell sub-clone (PC12-C41) with enhanced neurite outgrowth capacity: implications for a modulatory role of high molecular weight tau in neuritogenesis

Journal of Cell Science ◽

10.1242/jcs.106.2.611 ◽

1993 ◽

Vol 106 (2) ◽

pp. 611-626 ◽

Cited By ~ 4

Author(s):

K.K. Teng ◽

I.S. Georgieff ◽

J.M. Aletta ◽

J. Nunez ◽

M.L. Shelanski ◽

...

Keyword(s):

Molecular Weight ◽

Neurite Outgrowth ◽

Pc12 Cell ◽

Cell Types ◽

Neuronal Morphology ◽

Cytoskeletal Proteins ◽

Parental Line ◽

Pheochromocytoma Cells ◽

Tau Expression

To address the means by which diversity of neuronal morphology is generated, we have isolated and characterized naturally occurring variants of rat PC12 pheochromocytoma cells that exhibit altered neurite outgrowth properties in response to nerve growth factor (NGF). We describe here a PC12 cell sub-clone, designated PC12-clone 41 (PC12-C41), that displays significant increases in neurite abundance and stability when compared with the parental line. This difference does not appear to be due to an altered sensitivity or responsiveness to NGF or to a more rapid rate of neurite extension. Because of the role of the cytoskeleton in neuritogenesis, we examined a panel of the major cytoskeletal proteins (MAP 1.2/1B, beta-tubulin, chartins, peripherin, and high and low molecular weight (HMW and LMW) taus) whose levels and/or extent of phosphorylation are regulated by NGF in PC12 cultures. Although most cytoskeletal proteins showed little difference between PC12 and PC12-C41 cells (+/- NGF treatment), there was a significant contrast between the two lines with respect to tau expression. In particular, while NGF increases the total specific levels of tau in both cell types to similar extents (by about twofold), the proportion comprising HMW tau is threefold higher in the PC12-C41 clone than in PC12 cells. A comparable difference was observed under substratum conditions that were non-permissive for neurite outgrowth and so this effect was not merely a consequence of the differential neuritogenic capacities of the two lines. The distinction between the expression of HMW and LMW taus in PC12 and PC12-C41 cells (+/- NGF) was also observed at the level of the messages encoding these proteins. Such findings indicate that initiation of neurite outgrowth in PC12 cultures does not require a massive induction of tau expression and raise the possibility that HMW and LMW taus may have differential capacities for modulating neuronal morphology.

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Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase

Journal of Cell Science ◽

10.1242/jcs.109.2.289 ◽

1996 ◽

Vol 109 (2) ◽

pp. 289-300 ◽

Cited By ~ 3

Author(s):

T.R. Jackson ◽

I.J. Blader ◽

L.P. Hammonds-Odie ◽

C.R. Burga ◽

F. Cooke ◽

...

Keyword(s):

Neurite Outgrowth ◽

Pc12 Cells ◽

Specific Inhibitor ◽

Morphological Differentiation ◽

Sympathetic Neuron ◽

Neuronal Morphology ◽

Regulatory Subunit ◽

Membrane Reorganization ◽

Phosphoinositide 3 Kinase

Application of nerve growth factor (NGF) to PC12 cells stimulates a programme of physiological changes leading to the development of a sympathetic neuron like phenotype, one aspect of which is the development of a neuronal morphology characterised by the outgrowth of neuritic processes. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: firstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphological responses to NGF, the formation of actin filament rich microspikes and subsequent neurite outgrowth. This correlated with wortmannin inhibition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) and phosphatidylinositol(3,4)bisphosphate (PtdIns(3,4)P2) production and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the overexpression of a mutant p85 regulatory subunit of the phosphoinositide 3-kinase, which cannot interact with the catalytic p110 subunit, also substantially inhibited the initiation of NGF-stimulated neurite outgrowth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outgrowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP3, is a necessary second messenger for the cytoskeletal and membrane reorganization events which occur during neuronal differentiation.

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Interactions of a neuronal cell line (PC12) with laminin, collagen IV, and fibronectin: identification of integrin-related glycoproteins involved in attachment and process outgrowth.

The Journal of Cell Biology ◽

10.1083/jcb.105.5.2347 ◽

1987 ◽

Vol 105 (5) ◽

pp. 2347-2358 ◽

Cited By ~ 134

Author(s):

K J Tomaselli ◽

C H Damsky ◽

L F Reichardt

Keyword(s):

Cell Surface ◽

Cell Line ◽

Pc12 Cells ◽

Pc12 Cell ◽

Mammalian Cells ◽

Chinese Hamster ◽

Neuronal Cell ◽

Collagen Iv ◽

Nervous System Development ◽

Process Outgrowth

Neuronal responses to extracellular matrix (ECM) constituents are likely to play an important role in nervous system development and regeneration. We have studied the interactions of a neuron-like rat pheochromocytoma cell line, PC12, with ECM protein-coated substrates. Using a quantitative cell attachment assay, PC12 cells were shown to adhere readily to laminin (LN) or collagen IV (Col IV) but poorly to fibronectin (FN). The specificity of attachment to these ECM proteins was demonstrated using ligand-specific antibodies and synthetic peptides. To identify PC12 cell surface proteins that mediate interactions with LN, Col IV, and FN, two different antisera to putative ECM receptors purified from mammalian cells were tested for their effects on PC12 cell adhesion and neuritic process outgrowth. Antibodies to a 140-kD FN receptor heterodimer purified from Chinese hamster ovarian cells (anti-FNR; Brown, P. J., and R. L. Juliano, 1986, J. Cell Biol., 103:1595-1603) inhibited attachment to LN and FN but not to Col IV. Antibodies to an ECM receptor preparation purified from baby hamster kidney fibroblastic cells (anti-ECMR; Knudsen, K. A., P. E. Rao, C. H. Damsky, and C. A. Buck, 1981, Proc. Natl. Acad. Sci. USA., 78:6071-6075) inhibited attachment to LN, FN, and Col IV, but did not prevent attachment to other adhesive substrates. In addition to its effects on adhesion, the anti-ECMR serum inhibited both PC12 cell and sympathetic neuronal process outgrowth on LN substrates. Immunoprecipitation of surface-iodinated or [3H]glucosamine-labeled PC12 cells with either the anti-FNR or anti-ECMR serum identified three prominent cell surface glycoproteins of 120, 140, and 180 kD under nonreducing conditions. The 120-kD glycoprotein, which could be labeled with 32P-orthophosphate and appeared to be noncovalently associated with the 140- and 180-kD proteins, cross reacted with antibodies to the beta-subunit (band 3) of the avian integrin complex, itself a receptor or receptors for the ECM constituents LN, FN, and some collagens.

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Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.

The Journal of Cell Biology ◽

10.1083/jcb.126.3.801 ◽

1994 ◽

Vol 126 (3) ◽

pp. 801-810 ◽

Cited By ~ 435

Author(s):

K Jalink ◽

E J van Corven ◽

T Hengeveld ◽

N Morii ◽

S Narumiya ◽

...

Keyword(s):

Neurite Outgrowth ◽

Pc12 Cells ◽

Binding Protein ◽

Neuronal Cell ◽

Force Generation ◽

Gtp Binding Protein ◽

Adp Ribosylation ◽

Neurite Retraction ◽

Gtp Binding ◽

Shape Changes

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

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Estrogen Effects on Neurite Outgrowth and Cytoskeletal Gene Expression in ERα-Transfected PC12 Cell Lines

Experimental Neurology ◽

10.1006/exnr.2001.7744 ◽

2001 ◽

Vol 171 (2) ◽

pp. 308-316 ◽

Cited By ~ 24

Author(s):

Lakshmi Gollapudi ◽

Monica M. Oblinger

Keyword(s):

Gene Expression ◽

Neurite Outgrowth ◽

Cell Lines ◽

Pc12 Cell ◽

Estrogen Effects

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The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells.

The Journal of Cell Biology ◽

10.1083/jcb.102.3.821 ◽

1986 ◽

Vol 102 (3) ◽

pp. 821-829 ◽

Cited By ~ 122

Author(s):

C Richter-Landsberg ◽

B Jastorff

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Morphological Differentiation ◽

Intracellular Camp ◽

Nerve Growth ◽

Process Formation ◽

Dependent Protein

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.

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An investigation into the role of reactive oxygen species in the mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity using neuronal cell lines

Biochemical Pharmacology ◽

10.1016/0006-2952(93)90178-y ◽

1993 ◽

Vol 45 (4) ◽

pp. 927-933 ◽

Cited By ~ 20

Author(s):

Margaret Lai ◽

Helen Griffiths ◽

Hardev Pall ◽

Adrian Williams ◽

Joseph Lunec

Keyword(s):

Reactive Oxygen Species ◽

Cell Lines ◽

Neuronal Cell ◽

Reactive Oxygen ◽

Oxygen Species ◽

Neuronal Cell Lines

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Deciphering the Functional Role of RIPK4 in Melanoma

International Journal of Molecular Sciences ◽

10.3390/ijms222111504 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11504

Author(s):

Ewelina Madej ◽

Damian Ryszawy ◽

Anna A. Brożyna ◽

Malgorzata Czyz ◽

Jaroslaw Czyz ◽

...

Keyword(s):

Cell Lines ◽

Time Lapse ◽

Clinical Samples ◽

Invasive Potential ◽

Interacting Protein ◽

Metastatic Melanoma Cell ◽

Node Metastasis ◽

Different Levels

The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-1β level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-κB signaling in a RIPK4-dependent (RIPK4high) or independent (RIPK4low) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial).

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