Retinoic acid stimulates placental hormone secretion by choriocarcinoma cell lines in vitro.

Retinoic acid (RA), an active metabolite of vitamin A, is an important mediator of cellular differentiation and has been shown to stimulate human CG (hCG) secretion by JEG-3 choriocarcinoma cells in vitro. In order to determine whether RA stimulates the hCG secretion by other trophoblastic cell lines, we evaluated the effect of RA on hCG, hCG-alpha subunit (hCG-alpha), and progesterone secretion in three choriocarcinoma cell lines: JEG-3, JAR, and BeWo. RA stimulated hCG and hCG-alpha secretion in a dose-dependent fashion by each of the three cell lines. The time required to give a statistically significant increment of hCG and hCG-alpha over control cells was 48 h. The addition RA to cholera toxin (10 micrograms/ml) resulted in an additive or synergistic stimulation of hCG and hCG-alpha secretion by the three cell lines. Cycloheximide (1 microM) abolished the effect of RA on hCG and hCG-alpha secretion in the BeWo cell line. Progesterone secretion in response to RA was inconsistent. Progesterone secretion by both JEG-3 and BeWo cell lines were stimulated at high concentrations of RA (1 x 10(-6], whereas progesterone secretion by JAR cells was not stimulated. Intracellular levels of cAMP were not affected by RA treatment in the JEG and JAR cells. In the dosages used, RA did not significantly alter cell number in any of the cell lines. RA in physiologic concentrations stimulates hCG and hCG-alpha secretion by three choriocarcinoma cell lines in vitro. Whether RA is a physiologic mediator of placental hormone production is unknown.     

Thrombin modulates and reverses neuroblastoma neurite outgrowth.

Previous studies have shown that neuroblastoma cells and several types of primary neuronal cells in culture rapidly extend neurites when switched from serum-containing to serum-free medium. The present studies on cloned neuroblastoma cells show that thrombin blocked this spontaneous differentiation at 2 nM with a half-maximal potency of 50 pM. This required the catalytic activity of thrombin and was reversed upon thrombin removal. Thrombin also caused cells in serum-free medium to retract their neurites at equally low concentrations. Two other serine proteases, urokinase and plasmin, did not block or reverse neurite extension even at 100-fold higher concentrations. A specific assay for thrombin indicated that thrombin detected in serum-containing medium from neuroblastoma cultures was derived from serum and that it was likely responsible for much of the known capacity of serum to maintain neuroblastoma cells in a nondifferentiated state. This was supported by the finding that heparin addition reduced the thrombin concentration in serum-containing medium and stimulated neurite outgrowth from neuroblastoma cells in serum-containing medium. Studies on the ability of thrombin to modulate neurite outgrowth by other agents showed that it blocked and reversed the neurite outgrowth activity of two thrombin inhibitors: protease nexin-1 (which is identical to glial-derived neurite-promoting factor) and hirudin. Thrombin, however, did not block the neurite-promoting activity of dibutyryl cAMP or prostaglandin E1. These results suggest a specific role for thrombin in control of neurite outgrowth.     

Myelin-associated neurite growth-inhibitory proteins and suppression of regeneration of immature mammalian spinal cord in culture.

Neurite outgrowth across spinal cord lesions in vitro is rapid in preparations isolated from the neonatal opossum Monodelphis domestica up to the age of 12 days. At this age oligodendrocytes, myelin, and astrocytes develop and regeneration ceases to occur. The role of myelin-associated neurite growth-inhibitory proteins, which increase in concentration at 10-13 days, was investigated in culture by applying the antibody IN-1, which blocks their effects. In the presence of IN-1, 22 out of 39 preparations from animals aged 13-17 days showed clear outgrowth of processes into crushes. When 34 preparations from 13-day-old animals were crushed and cultured without antibody, no axons grew into the lesion. The success rate with IN-1 was comparable to that seen in younger animals but the outgrowth was less profuse. IN-1 was shown by immunocytochemistry to penetrate the spinal cord. Other antibodies which penetrated the 13-day cord failed to promote fiber outgrowth. To distinguish between regeneration by cut neurites and outgrowth by developing uncut neurites, fibers in the ventral fasciculus were prelabeled with carbocyanine dyes and subsequently injured. The presence of labeled fibers in the lesion indicated that IN-1 promoted regeneration. These results show that the development of myelin-associated growth-inhibitory proteins contributes to the loss of regeneration as the mammalian central nervous system matures. The definition of a critical period for regeneration, coupled with the ability to apply trophic as well as inhibitory molecules to the culture, can permit quantitative assessment of molecular interactions that promote spinal cord regeneration.     

Hot spots of retinoic acid synthesis in the developing spinal cord.

The embryonic spinal cord is known to be rich in retinoic acid, and several indirect lines of evidence point to a dorsoventral concentration difference of this compound. Previous measurements of dorsoventral retinoic acid levels, however, showed only minor differences. By a combination of microdissection and bioassay techniques, we compared retinoic acid levels with retinaldehyde dehydrogenase levels along spinal cords from early embryonic to postnatal mice. Both parameters vary in parallel, indicating that the principal reason for regional retinoic acid differences in the developing spinal cord is different levels of retinoic acid-generating enzyme. Consistent with previous reports, we observed overall quite high synthesis, decreasing with age, and no dorsoventral difference throughout much of the spinal cord length. In two locations, however, ventral synthesis exceeds dorsal synthesis by several orders of magnitude. These hot spots colocalize with the origins of the limb innervations. They are highest during early stages of limb innervation and disappear slowly postnatally. The synthesis hot spots are likely to create local retinoic acid diffusion halos, which may influence the survival of neurons in the limb regions of the spinal cord and which probably promote innervation of the developing limbs.     

An effect of nerve growth factor on parasympathetic neurite outgrowth.

Addition of nerve growth factor (NGF) to dissociated parasympathetic ciliary ganglion neurons resulted, within 60 min of its addition, in a 2-fold increase in average neurite length and an accompanying enlargement and spreading of neuronal growth cones. These effects occurred over a concentration range of NGF of 0.1-10 ng/ml and were blocked by affinity-purified antibody to NGF. Epidermal growth factor, fibroblast growth factor, and angiotensin did not have these effects, although insulin at high concentrations was able to induce a response similar to that of NGF. Dissociated sympathetic chain neurons also responded to NGF with increased neurite lengths, and, in addition, NGF considerably extended the survival time of these neurons in culture. However, the effect of NGF on ciliary ganglion neurons was limited to neurite outgrowth, and NGF did not promote the survival of these parasympathetic neurons.     

Protein kinase C mediates transient spinule-type neurite outgrowth in the retina during light adaptation.

Light and dark adaptation of the teleost retina is accompanied by a remarkable morphological rearrangement of the synaptic connections between photoreceptors and second-order neurons: during light adaptation, numerous new neurites, the so-called spinules, arise from the terminal dendrites of horizontal cells invaginating the cone pedicle, and during dark adaptation, these spinules are retracted. The formation of these spinules is paralleled by the appearance of color opponency in horizontal and ganglion cells, which led to the suggestion that these spinules are the site of the inhibitory synapses in the negative feedback loop between cones and horizontal cells. The formation of the spinules in the light and their disappearance in darkness have a time course of minutes and are modulated by the neurotransmitters dopamine and glutamate, respectively. Neurotransmitters can modulate neuronal processing through a variety of second messengers that activate protein kinases, resulting most commonly in protein phosphorylation. Herein we report that activation of protein kinase C by phorbol esters promotes the formation of new horizontal-cell spinules in animals kept in the dark. Partial inhibition of protein kinase C activation with sphingosines prevents the formation of new spinules during light adaptation but does not affect established spinules. The spinule-forming effect of phorbol esters is not mediated by dopaminergic neurons, since the effect is also seen in retinas depleted of dopaminergic neurons. Phorbol esters also initiate the formation of spinules in synaptically isolated horizontal cells, demonstrating that they have a direct action on these cells. In addition, isolated horizontal cells have substrate proteins that are phosphorylated in a protein kinase C-dependent manner.     

Peripheral-type benzodiazepines influence ornithine decarboxylase levels and neurite outgrowth in PC12 cells.

A number of the benzodiazepines (BZDs) inhibit nerve growth factor (NGF)-induced neurite outgrowth in a dose-dependent manner in PC12 cell cultures. The rank order of potency of a series of BZDs for inhibition of neurite outgrowth does not correlate with the order of their affinity constants for the so-called peripheral BZD sites present on PC12 cells. Whereas the inhibition of neurite extension is stereospecific, the binding to the peripheral site is not. The inhibition of neurite outgrowth is not attributable to a blockade of NGF binding to either its fast or slow receptors. Additionally, several other characteristics of NGF stimulation of PC12 cells, such as autoadhesion and the induction of ornithine decarboxylase (OrnDCase), remain unaltered in the presence of BZDs. Many BZDs increase OrnDCase levels in PC12 cells in the absence of NGF. The OrnDCase response to BZDs is blocked by actinomycin D. Furthermore, the structural requirements of BZDs for induction of OrnDCase activity is not identical to that for the inhibition of NGF-induced neurite extension. Thus, unlike attenuation of neurite extension, BZD induction of OrnDCase is not stereospecific. Also, some BZDs block neurite growth but do not induce OrnDCase. We propose that there are at least three sites of action of BZDs on PC12 cells. The first is the well-characterized high-affinity peripheral site. The second distinct locus of action results in a blockade of NGF-induced neurite outgrowth. The structural requirements for the latter effect are essentially indistinguishable from those for BZD induction of hemoglobin synthesis in Friend erythroleukemia cells. The third site of action results in an induction of OrnDCase. Structural requirements for this activity are not identical to those for either the differentiation of Friend cells or the inhibition of neurite outgrowth.     

Pituitary adenylate cyclase-activating polypeptide stimulates calcium mobilization in amphibian pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily. Although PACAP is a potent stimulator of adenylate cyclase activity in the adenohypophysis, the precise target cells for PACAP in the anterior pituitary remain unknown. The aim of the present study was to investigate whether PACAP could stimulate calcium mobilization in individual cells of the pituitary and to determine the type of cells that responded to PACAP. Enzymatically dispersed frog distal pituitary cells were plated on photoetched coverslips and cultured for 3-7 days. The cells were loaded with the fluorescent calcium indicator indo-1, and changes in intracellular calcium concentrations ([Ca2+]i) were monitored using dual wavelength microfluorimetry. The individual cells were localized with the aid of the alpha/numeric grid of the coverslips and identified retrospectively by immunofluorescence. Approximately 45% of GH and PRL cells and 25% of ACTH and TSH cells responded to PACAP (10(-5) M) ejection by an elevation of [Ca2+]i. Only 16% of gonadotropes were stimulated by PACAP. The time course of [Ca2+]i variations showed three different patterns: transient spikes, sustained stimulations, and oscillatory responses. In addition, heterogenous responses were observed within each cell type. These data provide evidence for the involvement of calcium mobilization in the mechanism of action of PACAP on pituitary cells. The results also indicate that in frogs, PACAP may stimulate the secretory activity of GH and PRL cells and, to a lesser extent, ACTH, TSH, and gonadotrope cells.     

Amyloid beta-protein as a substrate interacts with extracellular matrix to promote neurite outgrowth.

Progressive deposition of amyloid beta-protein (A beta) in brain parenchyma and blood vessels is a characteristic feature of Alzheimer disease. Recent evidence suggests that addition of solubilized synthetic A beta to medium may produce toxic or trophic effects on cultured hippocampal neurons. Because soluble A beta may not accumulate in significant quantities in brain, we asked whether immobilized A beta peptide as a substrate alters neurite outgrowth from cultured rat peripheral sensory neurons. This paradigm may closely mimic the conditions in Alzheimer disease brain tissue, in which neurites contact insoluble, extracellular aggregates of beta-amyloid. We detected no detrimental effects of A beta substrate on neurite outgrowth. Rather, A beta in combination with low doses of laminin or fibronectin enhanced neurite out-growth from these neuronal explants. Our results suggest that insoluble A beta in the cerebral neuropil may serve as a neurite-promoting matrix, perhaps explaining the apparent regenerative response of neurites observed around amyloid plaques in Alzheimer disease. Moreover, in concert with the recent discovery of A beta production by cultured neurons, our data suggest that A beta plays a normal physiological role in brain by complexing with the extracellular matrix.     

Sialidase enhances spinal axon outgrowth in vivo

The adult CNS is an inhibitory environment for axon outgrowth, severely limiting recovery from traumatic injury. This limitation is due, in part, to endogenous axon regeneration inhibitors (ARIs) that accumulate at CNS injury sites. ARIs include myelin-associated glycoprotein, Nogo, oligodendrocyte-myelin glycoprotein, and chondroitin sulfate proteoglycans (CSPGs). Some ARIs bind to specific receptors on the axon growth cone to halt outgrowth. Reversing or blocking the actions of ARIs may promote recovery after CNS injury. We report that treatment with sialidase, an enzyme that cleaves one class of axonal receptors for myelin-associated glycoprotein, enhances spinal axon outgrowth into implanted peripheral nerve grafts in a rat model of brachial plexus avulsion, a traumatic injury in which nerve roots are torn from the spinal cord. Repair using peripheral nerve grafts is a promising restorative surgical treatment in humans, although functional improvement remains limited. To model brachial plexus avulsion in the rat, C8 nerve roots were cut flush to the spinal cord and a peroneal nerve graft was inserted into the lateral spinal cord at the lesion site. Infusion of Clostridium perfringens sialidase to the injury site markedly increased the number of spinal axons that grew into the graft (2.6-fold). Chondroitinase ABC, an enzyme that cleaves a different ARI (CSPGs), also enhanced axon outgrowth in this model. In contrast, phosphatidylinositol-specific phospholipase C, which cleaves oligodendrocyte-myelin glycoprotein and Nogo receptors, was without benefit. Molecular therapies targeting sialoglycoconjugates and CSPGs may aid functional recovery after brachial plexus avulsion or other nervous system injuries and diseases.     

Retinoic acid induces alveolar regeneration in the adult mouse lung.

Recent data suggests that exogenous retinoic acid (RA) can induce alveolar regeneration in a mouse and a rat model of experimental emphysema and disrupted alveolar development. This may be because RA is required during normal alveolar development and the subsequent provision of RA reawakens the gene cascades used during development. Here, additional evidence that RA is required during alveologenesis in the mouse is provided by showing that disulphiram disrupts this process. A further model of disrupted alveolar development using dexamethasone administered postnatally is then described, and it is further shown that RA administered to these adult mice restores the lung architecture to normal. Alveolar regeneration with retinoic acid may therefore be an important novel therapeutic approach to the treatment of respiratory diseases characterised by a reduced gas-exchanging surface area, such as bronchopulmonary dysplasia and emphysema.     

Immunosuppressant FK506 promotes neurite outgrowth in cultures of PC12 cells and sensory ganglia.

The immunosuppressant drug FK506 acts by binding to receptor proteins, FK506-binding proteins (FKBPs), which in turn can bind to and regulate a Ca(2+)-dependent phosphatase, calcineurin, and a Ca2+ release channel, the ryanodine receptor. Based on our findings in regeneration models that levels of FKBPs during neural regeneration parallel those of growth-associated protein GAP43, a calcineurin substrate that regulates neurite extension, we examined effects of FK506 in PC12 rat pheochromocytoma cells and in rat sensory ganglia. FK506 enhances neurite outgrowth in both systems by increasing sensitivity to nerve growth factor. Blockade of FK506 actions in sensory ganglia by rapamycin, an FK506 antagonist, establishes that these effects involve FKBPs. Rapamycin itself stimulates neurite outgrowth in PC12 cells. These drug effects are detected at subnanomolar concentrations, suggesting therapeutic application in diseases involving neural degeneration.     

An astrocytic binding site for neuronal Thy-1 and its effect on neurite outgrowth.

Thy-1, a member of the immunoglobulin superfamily, is one of the most abundant glycoproteins on mammalian neurons. Nevertheless, its role in the peripheral or central nervous system is poorly understood. Certain monoclonal antibodies to Thy-1 promote neurite outgrowth by rodent central nervous system neurons in vitro, suggesting that Thy-1 functions, in part, by modulating neurite outgrowth. We describe a binding site for Thy-1 on astrocytes. This Thy-1-binding protein has been characterized by immunofluroesence with specific anti-idiotype monoclonal antibodies and by three competitive binding assays using (i) anti-idiotype antibodies, (ii) purified Thy-1, and (iii) Thy-1-transfected cells. The Thy-1-binding protein may participate in axonal or dendritic development in the nervous system.     

Proteolytic control of neurite outgrowth inhibitor NOGO-A by the cAMP/PKA pathway

Protein kinase A (PKA) controls major aspects of neurite outgrowth and morphogenesis and plays an essential role in synaptic plasticity and memory. However, the molecular mechanism(s) of PKA action on neurite sprouting and activity are still unknown. Here, we report that in response to neurotrophin or cAMP stimulation the RING ligase praja2 ubiquitinates and degrades NOGO-A, a major inhibitor of neurite outgrowth in mammalian brain. Genetic silencing of praja2 severely inhibited neurite extension of differentiating neuroblastoma cells and mesencephalic neurons and axon outgrowth and sprouting of striatal terminals in developing rat brain. This phenotype was rescued when both praja2 and NOGO-A were depleted, suggesting that NOGO-A is, indeed, a biologically relevant target of praja2 in neuronal cells. Our findings unveil a novel mechanism that functionally couples cAMP signaling with the proteolytic turnover of NOGO-A, positively impacting on neurite outgrowth in mammalian brain.Neurite outgrowth plays an essential role in embryonic development, neuronal differentiation, and central nervous system (CNS) plasticity. Outgrowth can be also altered in several neurological disorders, as well as by neuronal injury and degeneration (1, 2). Extracellular signals, such as neurotrophins (NTFs) and neurotransmitters, regulate neurite outgrowth, dendritic arborization, and synaptic activity, establishing a dynamic neuronal network in developing and adult CNS. NTFs and neurotransmitters act at the cell membrane by generating intracellular second messengers that, in turn, reversibly modulate the activity of signaling proteins and effector enzymes (3, 4).cAMP is an ancient second messenger that controls a variety of biological cues. In neurons, essential functions such as neurite outgrowth and morphogenesis, synaptic transmission, and plasticity require tightly regulated responses to cAMP/protein kinase A (PKA) stimulation (5). PKA holoenzyme localizes in subcellular microdomains through interactions with A-Kinase-Anchor-Proteins (AKAPs). AKAP forms a local transduction unit, which includes signaling/metabolic enzymes, receptors, ion channels, adaptor molecules, and mRNAs (6, 7). Space-restricted activation of PKA provides a control mechanism to direct, integrate, and locally attenuate the cAMP cascade (8). praja2 belongs to a growing family of mammalian RING ligases abundantly expressed in the brain that finely tune the stability of intracellular substrates and play an essential role in critical aspects of cell signaling. In response to cAMP stimulation, praja2 couples ubiquitination and proteolysis of the inhibitory PKA regulatory (R) subunits by the proteasome to a sustained cAMP/PKA signaling, significantly impacting on synaptic plasticity and long-term memory (9). In addition to enhancing cAMP signaling, the role of praja2 in neuronal differentiation and dendritic network in the CNS and the molecular targets involved are unknown.NOGO-A is a member of the reticulon (RTN) family of integral membrane proteins with a conserved C terminus reticulon homology domain (RHD) and abundantly expressed in oligodendrocytes and in distinct neuronal subpopulations (10, 11). NOGO-A was originally identified as a potent inhibitor of neurite outgrowth (1, 10). In the adult CNS and in injured neurons, NOGO-A restricts the capacity of an axon to grow and regenerate. Genetic ablation of NOGO-A promotes neuritogenesis and fasciculation of oligodendrocytes and culture of dorsal root ganglion neurons, functionally improving neuronal plasticity and recovery of postischemic adult rat brain (12).Although the role of NOGO-A in neurite outgrowth is well established, regulation of NOGO-A levels in differentiating neurons and the mechanism(s) involved have been, to date, unknown. Here, we report a novel mechanism of neuritogenesis based on proteolytic turnover of NOGO-A (13). In response to cAMP or neurothrophin stimulation, RING ligase praja2 ubiquitinates and degrades NOGO-A. Proteolysis of NOGO-A by praja2 is functionally linked to neurite outgrowth in both differentiating neurons and developing rat brain.     

Fibrinogen inhibits neurite outgrowth via beta 3 integrin-mediated phosphorylation of the EGF receptor

Changes in the molecular and cellular composition of the CNS after injury or disease result in the formation of an inhibitory environment that inhibits the regeneration of adult mammalian CNS neurons. Although a dramatic change in the CNS environment after traumatic injury or disease is hemorrhage because of vascular rupture or leakage of the blood-brain barrier, the potential role for blood proteins in repair processes remains unknown. Here we show that the blood protein fibrinogen is an inhibitor of neurite outgrowth that is massively deposited in the spinal cord after injury. We show that fibrinogen acts as a ligand for beta3 integrin and induces the transactivation of EGF receptor (EGFR) in neurons. Fibrinogen-mediated inhibition of neurite outgrowth is reversed by blocking either beta3 integrin or phoshorylation of EGFR. Inhibition of Src family kinases that mediate the cross-talk between integrin and growth factor receptors rescue the fibrinogen-induced phosphorylation of EGFR. These results identify fibrinogen as the first blood-derived inhibitor of neurite outgrowth and suggest fibrinogen-induced EGFR transactivation on neuronal cells as a molecular link between vascular and neuronal damage in the CNS after injury.     

Induction of neurite outgrowth by a conditioned-medium factor bound to the culture substratum

Heart-cell conditioned medium (HCM) induces rapid neurite outgrowth from isolated neurons in culture. The following evidence indicates that this action of HCM is due to a trypsin-sensitive factor which attaches to the polyornithinecoated culture substratum: (i) Pretreatment of the culture substratum with HCM allows rapid neurite outgrowth to occur even in unconditioned media. The active factor remains bound to the substratum during the period of neurite outgrowth. (ii) The substratum-bound activity is destroyed by trypsin treatment, but is insensitive to collagenase, RNase, and DNase. (iii) The factor that binds to the substratum is essential for neurite outgrowth, because HCM is no longer active when the material that binds to the polyornithine substratum has been removed by passage of the HCM over a series of culture dishes. However, this "depleted" HCM is still able to support the growth of nonneuronal cells. (iv) Most significantly, when neurons are cultured in whole HCM, the extent of neurite outgrowth is proportional to the amount of substratum-bound activity and not to the amount in solution, indicating that the substratum-bound form of the factor is more active. Previous observations [Collins, F. (1978) Dev. Biol. 65, 50-57] suggest that HCM promotes neurite outgrowth by increasing the adhesion between nerve cell surface extensions and the polyornithine-coated culture substratum. It is possible, therefore, that the factor in HCM that binds to the substratum possesses sites to which nerve cell surface components adhere.     

Retinoic acid redifferentiation therapy for thyroid cancer.

For the treatment of differentiated thyroid cancer, surgery, radioiodide therapy, and thyrotropin-suppressive thyroxine application represent established therapeutic measures of proven efficiency, affording a good prognosis for this disease. However, in up to 30% of the cases, dedifferentiation is observed, giving rise to tumors that are refractory to conventional treatment. Eventually, this may lead to the most malignant human tumor, anaplastic thyroid carcinoma, with a life expectancy of only a few months after diagnosis. Among novel approaches for the treatment of dedifferentiated thyroid carcinomas, retinoic acid redifferentiation therapy was evaluated in several in vitro and in vivo studies. Cell culture experiments in thyroid carcinoma lines show that RA treatment affects thyroid specific functions (type I 5'-deiodinase, sodium/iodide-symporter), cell-cell or cell-matrix interaction (intercellular adhesion molecule-1, E-cadherin), differentiation markers (alkaline phosphatase, CD97), growth, and tumorigenicity. The observed changes, which involve multiple parameters that characterize a mature, functional thyrocyte, may be interpreted as partial redifferentiation. In clinical pilot studies, about 40% of the patients responded to RA application with an increased radioiodide uptake. In an evaluation of 20 RA-treated patients with well-documented data sets, 8 exhibited a decrease (4) or stabilization (4) in tumor size and/or in serum thyroglobulin levels in addition to enhanced radioiodide transport. This indicates that these patients with a long history of unresponsiveness to other treatment may have experienced an actual therapeutic benefit. These data suggest that RA redifferentiation therapy, considering especially its comparatively mild side effects, may soon represent an alternative therapeutic approach to otherwise untreatable thyroid tumors.