Growth factor synergism and antagonism in early neural crest development.

This review article focuses on data that reveal the importance of synergistic and antagonistic effects in growth factor action during the early phases of neural crest development. Growth factors act in concert in different cell lineages and in several aspects of neural crest cell development, including survival, proliferation, and differentiation. Stem cell factor (SCF) is a survival factor for the neural crest stem cell. Its action is neutralized by neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) through apoptotic cell death. In contrast, SCF alone does not support the survival of melanogenic cells (pigment cell precursors). They require the additional presence of a neurotrophin (NGF, BDNF, or NT-3). Fibroblast growth factor-2 (FGF-2) is an important promoter of proliferation in neuronal progenitor cells. In neural crest cells, fibroblast growth factor treatment alone does not lead to cell expansion but also requires the presence of a neurotrophin. The proliferative stimulus of the fibroblast growth factor - neurotrophin combination is antagonized by transforming growth factor beta-1 (TGFbeta-1). Moreover, TGFbeta-1 promotes the concomitant expression of neuronal markers from two cell lineages, sympathetic neurons and primary sensory neurons, indicating that it acts on a pluripotent neuronal progenitor cell. Moreover, the combination of FGF-2 and NT3, but not other neurotrophins, promotes expression or activation of one of the earliest markers expressed by presumptive sympathetic neuroblasts, the norepinephrine transporter. Taken together, these data emphasize the importance of the concerted action of growth factors in neural crest development at different levels and in several cell lineages. The underlying mechanisms involve growth-factor-induced dependence of the cells on other factors and susceptibility to growth-factor-mediated apoptosis.     

Target-derived BMP signaling limits sensory neuron number and the extent of peripheral innervation in vivo

The role of target-derived BMP signaling in development of sensory ganglia and the sensory innervation of the skin was examined in transgenic animals that overexpress either the BMP inhibitor noggin or BMP4 under the control of a keratin 14 (K14) promoter. Overexpression of noggin resulted in a significant increase in the number of neurons in the trigeminal and dorsal root ganglia. Conversely, overexpression of BMP4 resulted in a significant decrease in the number of dorsal root ganglion neurons. There was no significant change in proliferation of trigeminal ganglion neurons in the noggin transgenic animals, and neuron numbers did not undergo the normal developmental decrease between E12.5 and the adult, suggesting that programmed cell death was decreased in these animals. The increase in neuron numbers in the K14-noggin animals was followed by an extraordinary increase in the density of innervation in the skin and a marked change in the pattern of innervation by different types of fibers. Conversely, the density of innervation of the skin was decreased in the BMP4 overexpressing animals. Further Merkel cells and their innervation were increased in the K14-noggin mice and decreased in the K14-BMP4 mice. The changes in neuron numbers and the density of innervation were not accompanied by a change in the levels of neurotrophins in the skin. These findings indicate that the normal developmental decrease in neuron numbers in sensory ganglia depends upon BMP signaling, and that BMPs may limit both the final neuron number in sensory ganglia as well as the extent of innervation of targets. Coupled with prior observations, this suggests that BMP signaling may regulate the acquisition of dependence of neurons on neurotrophins for survival, as well as their dependence on target-derived neurotrophins for determining the density of innervation of the target.     

Bone morphogenetic protein 4 (BMP4) signaling in retinoblastoma cells

Bone morphogenetic proteins (BMPs) - expressed in the developing retina - are known to be involved in the regulation of cell proliferation and apoptosis in several tumor entities. The objective of this study was to determine the role of the BMP4 pathway in retinoblastoma cells, which are absent in a functional retinoblastoma (RB1) gene. BMP receptors were detected in all retinoblastoma cell lines investigated. A correct transmission of BMP signaling via the Smad1/5/8 pathway could be demonstrated in WERI-Rb1 retinoblastoma cells and application of recombinant human BMP4 resulted in an increase in apoptosis, which to a large extend is caspase independent. Cell proliferation was not affected by BMP4 signaling, although the pRb-related proteins p107 and p130, contributing to the regulation of the same genes, are still expressed. WERI-Rb1 cells exhibit elevated endogenous levels of p21(CIP1) and p53, but we did not detect any increase in p53, p21(CIP1)or p27(KIP1) expression levels. Id proteins became, however, strongly up-regulated upon exogenous BMP4 treatment. Thus, RB1 loss in WERI-Rb1 cells is obviously not compensated for by pRb-independent (e.g. p53-dependent) cell cycle control mechanisms, preventing an anti-proliferative response to BMP4, which normally induces cell cycle arrest.     

Novel roles for neurotrophins are suggested by BDNF and NT-3 mRNA expression in developing neurons.

The results of our in situ hybridization experiments demonstrate that sensory neurons, sympathetic neurons, and motoneurons express brain-derived neurotrophic factor and/or neurotrophin-3 mRNAs during development in mouse. In accordance with previous data, we also find neurotrophins in the targets of sensory neurons (skin) and motoneurons (muscle) and the neurotrophin receptors p75, trkA, and trkB in sensory and sympathetic ganglia. These results suggest that neurotrophins have roles other than being target-derived factors that support neuron survival during developmental cell death (neurotrophic hypothesis), but may be transported in an orthograde fashion in neurons and released from axon terminals. We discuss several novel roles for neurotrophins, including autocrine/paracrine regulation of neuron survival, regulation of Schwann cell activity, and neuron to target signaling.     

Involvement of ras p21 in neurotrophin-induced response of sensory, but not sympathetic neurons

Little is known about the signal transduction mechanisms involved in the response to neurotrophins and other neurotrophic factors in neurons, beyond the activation of the tyrosine kinase activity of the neurotrophin receptors belonging to the trk family. We have previously shown that the introduction of the oncogene product ras p21 into the cytoplasm of chick embryonic neurons can reproduce the survival and neurite-outgrowth promoting effects of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and of ciliary neurotrophic factor (CNTF). To assess the potential signal- transducing role of endogenous ras p21, we introduced function-blocking anti-ras antibodies or their Fab fragments into cultured chick embryonic neurons. The BDNF-induced neurite outgrowth in E12 nodose ganglion neurons was reduced to below control levels, and the NGF- induced survival of E9 dorsal root ganglion (DRG) neurons was inhibited in a specific and dose-dependent fashion. Both effects could be reversed by saturating the epitope-binding sites with biologically inactive ras p21 before microinjection. Surprisingly, ras p21 did not promote the survival of NGF-dependent E12 chick sympathetic neurons, and the NGF-induced survival in these cells was not inhibited by the Fab-fragments. The survival effect of CNTF on ras-responsive ciliary neurons could not be blocked by anti-ras Fab fragments. These results indicate an involvement of ras p21 in the signal transduction of neurotrophic factors in sensory, but not sympathetic or ciliary neurons, pointing to the existence of different signaling pathways not only in CNTF-responsive, but also in neurotrophin-responsive neuronal populations.     

Multiple effects of artemin on sympathetic neurone generation, survival and growth

To define the role of artemin in sympathetic neurone development, we have studied the effect of artemin on the generation, survival and growth of sympathetic neurones in low-density dissociated cultures of mouse cervical and thoracic paravertebral sympathetic ganglia at stages throughout embryonic and postnatal development. Artemin promoted the proliferation of sympathetic neuroblasts and increased the generation of new neurones in cultures established from E12 to E14 ganglia. Artemin also exerted a transient survival-promoting action on newly generated neurones during these early stages of development. Between E16 and P8, artemin exerted no effect on survival, but by P12, as sympathetic neurones begin to acquire neurotrophic factor independent survival, artemin once again enhanced survival, and by P20 it promoted survival as effectively as nerve growth factor (NGF). During this late period of development, artemin also enhanced the growth of neurites from cultured neurones more effectively than NGF. Confirming the physiological relevance of the mitogenic action of artemin on cultured neuroblasts, there was a marked reduction in the rate of neuroblast proliferation in the sympathetic ganglia of mice lacking the GFRalpha3 subunit of the artemin receptor. These results indicate that artemin exerts several distinct effects on the generation, survival and growth of sympathetic neurones at different stages of development.     

Functional roles of neurotrophin 3 in the developing and mature sympathetic nervous system

Nerve growth factor (NGF) is a potent regulator of sympathetic neuronal function in both developing and adult animals. This article reviews the evidence published in recent years indicating that another member of the NGF family, neurotrophin 3 (NT3), plays both a complementary and overlapping role in the development and maturation of sympathetic neurons. In migratory neural crest cells, expression of the high-affinity receptor, trkC, and promotion of mitosis by NT3 suggest an involvement in gangliogenesis, since sympathetic neuroblasts express both NT3 and trkC and require NT3 for their proliferation, differentiation, and survival, it has been proposed that the factor acts at this developmental stage as an autocrine or paracrine factor. However, NT3 also acts in parallel with NGF to promote the survival of postmitotic neurons during late development. Both trkC and trkA are expressed in sympathetic neurons and function as high-affinity receptors for NT3. NT3 is synthesized in sympathetic effector tissues and the endogenous factor is retrogradely transported to accumulate within the cell soma. Thus, in addition to its role in the differentiation of sympathetic neurons, NT3, like NGF, is also an effector tissue-derived neurotrophic factor for these neurons in maturity.     

Neurotrophins and cell death

The neurotrophins - NGF, BDNF, NT-3 - are secreted proteins that play a major role in neuron survival, differentiation and axon wiring toward target territories. They do so by interacting with their main tyrosine kinase receptors TrkA, TrkB, TrkC and p75(NTR). Even though there is a general consensus on the view that neurotrophins are survival factors, there are two fundamentally different views on how they achieve this survival activity. One prevailing view is that all neurons and more generally all normal cells are naturally committed to die unless a survival factor blocks this death. This death results from the engagement of a "default" apoptotic cell program. The minority report supports, on the opposite, that neurotrophin withdrawal is associated with an active signal of cell death induced by unbound dependence receptors. We will discuss here how neurotrophins regulate cell death and survival and how this has implications not only during nervous system development but also during cancer progression.     

BMP2 and BMP6 control p57(Kip2) expression and cell growth arrest/terminal differentiation in normal primary human epidermal keratinocytes

Functional studies of the canonical Bone Morphogenetic Protein (BMP) signalling pathway in human epidermal keratinocytes have been limited to the immortalized and p53-mutated HaCaT cells and are primarily dependent on BMP6 treatment in mouse epidermal keratinocytes. Despite these insightful analyses, the molecular mechanism underlying the role of BMP signalling in the precise balance between growth arrest and terminal differentiation of keratinocytes still remains not clearly defined. The current study first investigated the hitherto uncharacterized status and functions of BMP signalling in normal human keratinocytes by using three independent strains of primary interfollicular epidermal keratinocytes. Then we provided data demonstrating the role of BMP2 compared to BMP6 in the inhibition of growth and induction of subsequent terminal differentiation of these cells. A second relevant finding is based on the clonal analysis of colony types present in untreated and BMP2/6-treated cultures in absence of EGF. BMP treatment results in the clonal transition from proliferative to abortive colonies, suggesting that BMP signalling most likely inhibits stem cell proliferation and triggers cell cycle exit from transit amplifying cells. Third, we showed evidence that, of the three members of the Cip/Kip family of cyclin-dependent kinase inhibitors, only p57(Kip2) and p21(Cip1) have a BMP2/6-induced expression. One mechanism of inhibition of cell proliferation involves p57(Kip2) as an immediate early response, in contradistinction with p21(Cip1) which largely depends on de novo protein synthesis for its effect to proceed. All together, these results clarify the BMP signalling status in normal primary human keratinocytes and support a new mechanism of inhibition of the proliferation of interfollicular epidermal keratinocytes coupled with induction of their terminal differentiation following BMP2 or BMP6 addition.     

TAM Receptors Support Neural Stem Cell Survival,Proliferation and Neuronal Differentiation

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III⁺) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.     

Neurotrophins mediate human embryonic stem cell survival

Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family, which mediate antiapoptotic signals. We show that three TRK ligands, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins, TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation.     

Interplay between BMP4 and IL-7 in human intrathymic precursor cells

Bone morphogenetic proteins (BMPs) play a pivotal role during vertebrate embryogenesis and organogenesis, and have also been described to function in regulating cell fate and determination in self-renewing tissues in adults. Recent results have demonstrated that the different components of the BMP2/4 signaling pathway are expressed in the human thymus. In this study, we provide evidence that BMP4 and IL-7 interplay is important in the maintenance of the human thymic progenitor population. Intrathymic CD34⁺ cells express BMP receptors (BMPRIA, BMPRIB, ActRIA, BMPRII), signal transduction molecules (Smad1, 5, 8 and 4), and produce BMP4. Neutralization of endogenous BMP4 by treatment with the antagonist Noggin reduces thymic precursor cell survival, and the addition of exogenous BMP4 decreases their proliferation. The treatment of chimeric human-mouse fetal thymus organ cultures with BMP4 inhibits cell expansion, arrests thymocyte differentiation, and leads to the accumulation of human CD34⁺ precursor cells. This effect is mainly attributed to the ability of BMP4 to counteract the IL-7-induced proliferation and differentiation of CD34⁺ cells. BMP4 down-regulates in the precursor cell population the expression of CD127 and inhibits the IL-7-dependent STAT5 phosphorylation. In addition, BMP signaling is promoted by IL-7. Our results also demonstrate that in thymic progenitors BMPs act downstream of Sonic Hedgehog, previously described to function as a maintenance factor for human intrathymic CD34⁺ precursor cells.     

Bone Morphogenetic Protein-2 Promotes Survival and Differentiation of Striatal GABAergic Neurons in the Absence of Glial Cell Proliferation

We examined the potential neurotrophic effects of bone morphogenetic protein (BMP)-2 on the survival and differentiation of neurons cultured from the rat developing striatum at embryonic day 16, a period during which the mRNAs for BMP-2 and its receptor subunits (types IA, IB, and II) were detected. BMP-2 exerted potent activity to promote the survival of striatal neurons and increased the number of surviving microtubule-associated protein-2-positive cells by 2.4-fold as compared with the control cultures after 4 days in vitro. Although basic fibroblast growth factor (bFGF) also showed relatively high activity to promote the survival of striatal neurons, transforming growth factor-beta1, -beta2, and -beta3, glial cell line-derived neurotrophic factor, or brain-derived neurotrophic factor promoted their survival weakly. Striatal neurons cultured in the presence of BMP-2 or bFGF possessed extensive neurite outgrowths, the majority of which were GABA-immunoreactive. Inhibition of glial cell proliferation by 5-fluorodeoxyuridine did not affect the capacity of BMP-2 to promote the survival of striatal GABAergic neurons. In contrast, the ability of bFGF to promote the survival of striatal neurons was inhibited significantly by the treatment of cells with 5-fluorodeoxyuridine. All these results suggest that BMP-2 exerts potent neurotrophic effects on the striatal GABAergic neurons in a glial cell-independent manner.     

Nerve growth factor, neural stem cells and Alzheimer's disease

The protein family of the neurotrophins (NTs) comprises structurally and functionally related molecules such as nerve growth factor (NGF) which influences the proliferation, differentiation, survival and death of neuronal cells. In addition to their established functions for cell survival, NTs also mediate higher brain activities such as learning and memory. Changes in NT expression levels have thus been implicated in neurological diseases such as Alzheimer's disease (AD), an age-related neurodegenerative disorder that is characterized by progressive loss of memory and deterioration of higher cognitive functions. The present review provides an overview of the functional role of NGF in neural stem cells and AD while pointing to a potential application of this peptide for the treatment of AD.