Neuronal and glial localization of GABA transporter immunoreactivity in the myenteric plexus

The neurotransmitter gamma-aminobutyric acid (GABA) is removed from the extracellular space by sodium and chloride dependent high affinity plasma membrane transporters. In the rat central nervous system, three GABA transporters, GAT1, GAT2 and GAT3, have been cloned and localized by immunohistochemistry. The purpose of this study was to examine the distribution of these transporters within the myenteric plexus of the rat gastrointestinal tract. We investigated their cellular locations using GAT1-3 specific antisera in lightly fixed segments of rat duodenum, ileum and colon. Immunohistochemistry revealed a large number of GAT2-immunoreactive structures that surrounded neurons within each ganglion of the myenteric plexus. GAT2 was colocalized in these structures with the glial cell marker p75(NTR), suggesting that the predominant high affinity GABA transporter within enteric glia is GAT2. GAT3 immunoreactivity was localized within many nerve cell bodies, and no labeling for GAT1 was detected, although it was present in retina, which was used as a control. Double labeling for calretinin and nitric oxide synthase (NOS) revealed colocalization of GAT3 with approximately 75% of calretinin-immunoreactive neurons and 15% of NOS-immunoreactive neurons. This suggests that a small proportion of inhibitory motor neurons and at least some putative intrinsic primary afferent neurons within the rat gastrointestinal tract express GAT3. Thus NOS neurons, which appear to utilize GABA as a transmitter, and calretinin-immunoreactive neurons, which do not appear to be GABAergic, both express immunoreactivity for GABA transporters.     

Developmental Expression of GABA Transporters GAT1 and GAT4 Suggests Involvement in Brain Maturation

Abstract: cDNA clones representing four pharmacologically distinct GABA transporters (GAT1–GAT4) were previously identified in mouse brain. Two of these, GAT1 and GAT4, were found to be brain specific. We studied GAT1 and GAT4 in the developing rat brain using polyclonal antibodies against recombinant fusion proteins. Patterns of immunoreactivity were very similar in the embryonic and early postnatal stages for both transporters. However, whereas GAT1 immunoreactivity was detected in distinct patterns in gray matter and growing axons, GAT4 immunoreactivity was found in a subset of radial glial cell fascicles. These patterns usually oriented perpendicularly to the axons expressing GAT1. Our results suggest a transient relationship between GAT4-expressing radial glial elements and GAT1-expressing axons. The presence of GAT1 in the cortical marginal zone and the numerous GAT4-positive fascicles observed in the fetal anterior commissure indicate that both transporters may play a role in processes of brain maturation. Because the beginning of expression for both GAT1 and GAT4 correlates with the expression of the α1 subunit of the GABA receptor, the transporters may be connected with the maturation of adult-type GABAergic inhibitory system in the brain.     

A Possible Role of the Non-GAT1 GABA Transporters in Transfer of GABA From GABAergic to Glutamatergic Neurons in Mouse Cerebellar Neuronal Cultures

Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, β-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC₅₀ 142 μM), β-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC₅₀ 0.8 μM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 μM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of β-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also GAT2 or GAT3 (HUGO nomenclature) could play a role.     

Overexpression of γ－aminobutyric acid transporter subtype I leads to susceptibility to kainic acid－induced seizure in transgenic mice

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABA(A) receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.     

The Caenorhabditis elegans snf-11 gene encodes a sodium-dependent GABA transporter required for clearance of synaptic GABA

Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in Caenorhabditis elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na+ and Cl- dependent, has an EC50 value of 168 microM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or postsynaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans and that the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.     

GABA Transporter-1 Deficiency Confers Schizophrenia-Like Behavioral Phenotypes

The mechanism underlying the pathogenesis of schizophrenia remains poorly understood. The hyper-dopamine and hypo-NMDA receptor hypotheses have been the most enduring ideas. Recently, emerging evidence implicates alterations of the major inhibitory system, GABAergic neurotransmission in the schizophrenic patients. However, the pathophysiological role of GABAergic system in schizophrenia still remains dubious. In this study, we took advantage of GABA transporter 1 (GAT1) knockout (KO) mouse, a unique animal model with elevated ambient GABA, to study the schizophrenia-related behavioral abnormalities. We found that GAT1 KO mice displayed multiple behavioral abnormalities related to schizophrenic positive, negative and cognitive symptoms. Moreover, GAT1 deficiency did not change the striatal dopamine levels, but significantly enhanced the tonic GABA currents in prefrontal cortex. The GABA_A receptor antagonist picrotoxin could effectively ameliorate several behavioral defects of GAT1 KO mice. These results identified a novel function of GAT1, and indicated that the elevated ambient GABA contributed critically to the pathogenesis of schizophrenia. Furthermore, several commonly used antipsychotic drugs were effective in treating the locomotor hyperactivity in GAT1 KO mice, suggesting the utility of GAT1 KO mice as an alternative animal model for studying schizophrenia pathogenesis and developing new antipsychotic drugs.     

Role of microRNA‐155 in modifying neuroinflammation and γ‐aminobutyric acid transporters in specific central regions after post‐ischaemic seizures

In the central nervous system, interleukin (IL)‐1β, IL‐6 and tumour necrosis factor (TNF)‐α have a regulatory role in pathophysiological processes of epilepsy. In addition, γ‐aminobutyric acid (GABA) transporter type 1 and type 3 (GAT‐1 and GAT‐3) modulate the levels of extracellular GABA in involvement in the neuroinflammation on epileptogenesis. Thus, in the current report we examined the effects of inhibiting microRNA‐155 (miR‐155) on the levels of IL‐1β, IL‐6 and TNF‐α, and expression of GAT‐1 and GAT‐3 in the parietal cortex, hippocampus and amygdala of rats with nonconvulsive seizure (NCS) following cerebral ischaemia. Real time RT‐PCR, ELISA and Western blot analysis were used to examine the miR‐155, proinflammatory cytokines (PICs) and GAT‐1/GAT‐3 respectively. With induction of NCS, the levels of miR‐155 were amplified in the parietal cortex, hippocampus and amygdala and this was accompanied with increases of IL‐1β, IL‐6 and TNF‐α. In those central areas, expression of GAT‐1 and GAT‐3 was upregulated; and GABA was reduced in rats following NCS. Intracerebroventricular infusion of miR‐155 inhibitor attenuated the elevation of PICs, amplification of GAT‐1 and GAT‐3 and impairment of GABA. Furthermore, inhibition of miR‐155 decreased the number of NCS events following cerebral ischaemia. Inhibition of miR‐155 further improved post‐ischaemia‐evoked NCS by altering neuroinflammation‐GABA signal pathways in the parietal cortex, hippocampus and amygdala. Results suggest the role of miR‐155 in regulating post‐ischaemic seizures via PICs‐GABA mechanisms.     

Early morphological and functional changes in the GABAergic system of hippocampus in the rat lithium-pilocarpine model of epilepsy

We studied early alterations in the GABAergic system of the rat hippocampus in the lithium–pilocarpine model of epilepsy. Twenty-four hours after the pilocarpine treatment, a decrease in the number of calretinin-positive interneurons was observed in the CA1 field of the hippocampus, whereas the number of parvalbumin-positive interneurons remained unchanged. The decreased levels of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD67) and the membrane GABA transporter GAT1 were revealed using Western blot analysis. These data indicate an altered excitation/inhibition balance in the hippocampus with excitation dominance.     

Functional expression of release-regulating glycine transporters GLYT1 on GABAergic neurons and GLYT2 on astrocytes in mouse spinal cord

It is widely accepted that glycine transporters of the GLYT1 type are situated on astrocytes whereas GLYT2 are present on glycinergic neuronal terminals where they mediate glycine uptake. We here used purified preparations of mouse spinal cord nerve terminals (synaptosomes) and of astrocyte-derived subcellular particles (gliosomes) to characterize functionally and morphologically the glial versus neuronal distribution of GLYT1 and GLYT2. Both gliosomes and synaptosomes accumulated [3H]GABA through GAT1 transporters and, when exposed to glycine in superfusion conditions, they released the radioactive amino acid not in a receptor-dependent manner, but as a consequence of glycine penetration through selective transporters. The glycine-evoked release of [3H]GABA was exocytotic from synaptosomes but GAT1 carrier-mediated from gliosomes. Based on the sensitivity of the glycine effects to selective GLYT1 and GLYT2 blockers, the two transporters contributed equally to evoke [3H]GABA release from GABAergic synaptosomes; even more surprising, the 'neuronal' GLYT2 contributed more efficiently than the 'glial' GLYT1 to mediate the glycine effect in [3H]GABA releasing gliosomes. These functional results were largely confirmed by confocal microscopy analysis showing co-expression of GAT1 and GLYT2 in GFAP-positive gliosomes and of GAT1 and GLYT1 in MAP2-positive synaptosomes. To conclude, functional GLYT1 are present on neuronal axon terminals and functional GLYT2 are expressed on astrocytes, indicating not complete selectivity of glycine transporters in their glial versus neuronal localization in the spinal cord.     

The molecular mechanism underlying GABAergic dysfunction in nucleus accumbens of depression‐like behaviours in mice

Depression is the most frequent psychiatric disorder in the world. Recent evidence has shown that stress‐induced GABAergic dysfunction in the nucleus accumbens (NAc) contributed to the pathophysiology of depression. However, the molecular mechanisms underlying these pathological changes remain unclear. In this study, mice were constantly treated with the chronic unpredictable mild stress (CUMS) till showing depression‐like behaviours expression. GABA synthesis, release and uptake in the NAc tissue were assessed by analysing the expression level of genes and proteins of Gad‐1, VGAT and GAT‐3 by qRT‐PCR and Western blotting. The miRNA/mRNA network regulating GABA was constructed based on the bioinformatics prediction software and further validated by dual‐luciferase reporter assay in vitro and qRT‐PCR in vivo, respectively. Our results showed that the expression level of GAT‐3, Gad‐1 and VGAT mRNA and protein significantly decreased in the NAc tissue from CUMS‐induced depression‐like mice than that of control mice. However, miRNA‐144‐3p, miRNA‐879‐5p, miR‐15b‐5p and miRNA‐582‐5p that directly down‐regulated the expression of Gad‐1, VGAT and GAT‐3 were increased. In the mRNA/miRNA regulatory GABA network, Gad‐1 and VGAT were directly regulated by binding seed sequence of miR‐144‐3p, and miR‐15b‐5p, miR‐879‐5p could be served negative post‐regulators by binding to the different sites of VGAT 3′‐UTR. Chronic stress causes the impaired GABA synthesis, release and uptake by up‐regulating miRNAs and down‐regulating mRNAs and proteins, which may reveal the molecular mechanisms for the decreased GABA concentrations in the NAc tissue of CUMS‐induced depression.     

GABA transporters inDrosophila melanogaster: molecular cloning, behavior, and physiology

Molecular cloning of GABA transporter-homologous cDNAs from aDrosophila melanogaster headspecific library was accomplished using a conserved oligomer from a highly conserved domain within the mammalian GABA transporters. Partial DNA sequencing of these cDNAs demonstrated homology with the mammalian transporters, indicating these are ancient, evolutionarily conserved molecules. Although theDrosophila cDNAs had distinct restriction enzyme patterns, they recognized the same locus inDrosophila genomic DNA, suggesting that the multiple isoforms might arise via alternative splicing. Antibodies specific for the mammalian GABA transporters GAT1, GAT2 and GAT3 recognized non-overlapping and developmentally distinct patterns of expression inDrosophila neuronal tissues. Treatment of larval instars with nipecotic acid, a generalized GABA reuptake inhibitor, revealed specific, dose-dependent alterations in behavior consistent with the presence of multiple transporter molecules with differing affinities for this drug. Synaptic current recordings revealed that nipecotic acid treated larvae have an increase in latency jitter of evoked quantal release, resulting in a broader average excitatory junctional current which was manifested in a broader EJP. These results imply that alterations in the development of the CNS occur if GABAergic neurotransmission is protentiated during development. The data suggest that, as in mammals, there are multiple GABA transporters inDrosophila whose expression is differentially regulated.     

Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter I （GAT1）

It is well documented that 7-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABAA and GABAB receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter I (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wild-type mice. Our findings provided the first evidence that abnormal expression of GAT1 could result in dysgenesis,and indicated that GAT1 might be therapeutically targeted for contraception or dysgenesis treatment.     

Blocking the GABA transporter GAT‐1 ameliorates spinal GABAergic disinhibition and neuropathic pain induced by paclitaxel

Paclitaxel is a chemotherapeutic agent widely used for treating carcinomas. Patients receiving paclitaxel often develop neuropathic pain and have a reduced quality of life which hinders the use of this life‐saving drug. In this study, we determined the role of GABA transporters in the genesis of paclitaxel‐induced neuropathic pain using behavioral tests, electrophysiology, and biochemical techniques. We found that tonic GABA receptor activities in the spinal dorsal horn were reduced in rats with neuropathic pain induced by paclitaxel. In normal controls, tonic GABA receptor activities were mainly controlled by the GABA transporter GAT‐1 but not GAT‐3. In the spinal dorsal horn, GAT‐1 was expressed at presynaptic terminals and astrocytes while GAT‐3 was only expressed in astrocytes. In rats with paclitaxel‐induced neuropathic pain, the protein expression of GAT‐1 was increased while GAT‐3 was decreased. This was concurrently associated with an increase in global GABA uptake. The paclitaxel‐induced attenuation of GABAergic tonic inhibition was ameliorated by blocking GAT‐1 but not GAT‐3 transporters. Paclitaxel‐induced neuropathic pain was significantly attenuated by the intrathecal injection of a GAT‐1 inhibitor. These findings suggest that targeting GAT‐1 transporters for reversing disinhibition in the spinal dorsal horn may be a useful approach for treating paclitaxel‐induced neuropathic pain.

     

Novel Properties of a Mouse γ-Aminobutyric Acid Transporter (GAT4)

We expressed the mouse gamma-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/ human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/ Cl-/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q10) of 4.3. The GAT4 turnover rate (1.5 s(-l); -50 mV, 21 degrees C) and temperature dependence suggest physiological turnover rates of 15-20 s(-1). No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl- leak current (ICl(leak)) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (ICl(leak)) followed the Cl- Nernst potential. (ii) A Li+ leak current (ILi(leak)) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q10 approximately 1). The two leak modes appeared not to coexist, as Li+ inhibited (ICl(leak)). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/Cl-/GABA cotransport, ILi(leak), and ICl(leak), (Ki approximately 30 microM), and the voltage-induced presteady-state charge movements (Ki approximately 440 microM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jicroumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.     

Selective inhibitors of GABA uptake: synthesis and molecular pharmacology of 4-N-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol analogues

A series of lipophilic diaromatic derivatives of the glia-selective GABA uptake inhibitor (R)-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol [(R)-exo-THPO, 4] were synthesized via reductive amination of 3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazol-4-one (9) or via N-alkylation of O-alkylatedracemic 4. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation or primary cultures of mouse cortical neurons and glia cells (astrocytes), as well as HEK cells transfected with cloned mouse GABA transporter subtypes (GAT1-4). The activity against isoniazid-induced convulsions in mice after subcutaneous administration of the compounds was determined. All of the compounds were potent inhibitors of synaptosomal uptake the most potent compound being (RS)-4-[N-(1,1-diphenylbut-1-en-4-yl)amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (17a, IC50 = 0.14 microM). The majority of the compounds showed a weak preference for glial, as compared to neuronal, GABA uptake. The highest degree of selectivity was 10-fold corresponding to the glia selectivity of (R)-N-methyl-exo-THPO (5). All derivatives showed a preference for the GAT1 transporter, as compared with GAT2-4, with the exception of (RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (28d), which quite surprisingly turned out to be more potent than GABA at both GAT1 and GAT2 subtypes. The GAT1 activity was shown to reside in (R)-28d whereas (R)-28d and (S)-28d contributed equally to GAT2 activity. This makes (S)-28d a GAT2 selective compound, and (R)-28d equally effective in inhibition of GAT1 and GAT2 mediated GABA transport. All compounds tested were effective as anticonvulsant reflecting that these compounds have blood-brain barrier permeating ability.     

A novel selective gamma-aminobutyric acid transport inhibitor demonstrates a functional role for GABA transporter subtype GAT2/BGT-1 in the CNS

The system of GABA transporters in neural cells constitutes an efficient mechanism for terminating inhibitory GABAergic neurotransmission. This transport system is an important therapeutical target in epileptic disorders, but potentially also in other neurological disorders. Thus, selective intervention in GABA uptake has been the subject of extensive research for several decades. In a series of lipophilic diaromatic derivatives of (RS)-3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO), N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) turned out to be an equipotent inhibitor at the mouse transporters GAT1 and GAT2 (BGT-1) but inactive at GAT3 and GAT4. This novel pharmacological profile among GABA uptake inhibitors prompted a thorough investigation of the in vivo properties of this compound. These investigations have for the first time demonstrated a functional role for GABA transporter subtype GAT2/BGT-1, which points to the therapeutic relevance of inhibiting this transporter subtype. An overview of the development and characterisation of EF1502 is presented here.     

Functional regulation of gamma-aminobutyric acid transporters by direct tyrosine phosphorylation

Tyrosine phosphorylation regulates multiple cell signaling pathways and functionally modulates a number of ion channels and receptors. Neurotransmitter transporters, which act to clear transmitter from the synaptic cleft, are regulated by multiple second messenger pathways that exert their effects, at least in part, by causing a redistribution of the transporter protein to or from the cell surface. To test the hypothesis that tyrosine phosphorylation affects transporter function and to determine its mechanism of action, we examined the regulation of the rat brain gamma-aminobutyric acid (GABA) transporter GAT1 expressed endogenously in hippocampal neurons and expressed heterologously in Chinese hamster ovary cells. Inhibitors of tyrosine kinases decreased GABA uptake; inhibitors of tyrosine phosphatases increased GABA uptake. The decrease in uptake seen with tyrosine kinase inhibitors was correlated with a decrease in tyrosine phosphorylation of GAT1 and resulted in a redistribution of the transporter from the cell surface to intracellular locations. A mutant GAT1 construct that was refractory to tyrosine phosphorylation could not be regulated by tyrosine kinase inhibitors. Activators of protein kinase C, which are known to cause a redistribution of GAT1 from the cell surface, were additive to the effects of tyrosine kinase inhibitors suggesting that multiple signaling pathways control transporter redistribution. Application of brain-derived neurotrophic factor, which activates receptor tyrosine kinases, up-regulated GAT1 function suggesting one potential trigger for the cellular regulation of GAT1 signaling by tyrosine phosphorylation. These data support the hypothesis that transporter expression and function is controlled by the interplay of multiple cell signaling cascades.