The beta-PDGF receptor induces neuronal differentiation of PC12 cells.

Expression of the mouse beta-PDGF receptor by gene transfer confers PDGF-dependent and reversible neuronal differentiation of PC12 pheochromocytoma cells similar to that observed in response to NGF and basic FGF. A common property of the PDGF, NGF, and basic FGF-induced differentiation response is the requirement for constant exposure of cells to the growth factor. To test the hypothesis that a persistent level of growth factor receptor signaling is required for the maintenance of the neuronal phenotype, we examined the regulation of the serine/threonine-specific MAP kinases after either short- (10 min) or long-term (24 h) stimulation with growth factors. Mono Q FPLC resolved two peaks of growth factor-stimulated MAP kinase activity that coeluted with tyrosine phosphorylated 41- and 43-kDa polypeptides. MAP kinase activity was markedly stimulated (approximately 30-fold) within 5 min of exposure to several growth factors (PDGF, NGF, basic FGF, EGF, and IGF-I), but was persistently maintained at 10-fold above basal activity after 24 h only by the growth factors that also induce PC12 cell differentiation (PDGF, NGF, and basic FGF). Thus the beta-PDGF receptor is in a subset of tyrosine kinase-encoded growth factor receptors that are capable of maintaining continuous signals required for differentiation of PC12 cells. These signals include the constitutive activation of cytoplasmic serine/threonine protein kinases.     

Rab7 mutants associated with Charcot-Marie-Tooth disease exhibit enhanced NGF-stimulated signaling

Missense mutants in the late endosomal Rab7 GTPase cause the autosomal dominant peripheral neuropathy Charcot-Marie-Tooth disease type 2B (CMT2B). As yet, the pathological mechanisms connecting mutant Rab7 protein expression to altered neuronal function are undefined. Here, we analyze the effects Rab7 CMT2B mutants on nerve growth factor (NGF) dependent intracellular signaling in PC12 cells. The nerve growth factor receptor TrkA interacted similarly with Rab7 wild-type and CMT2B mutant proteins, but the mutant proteins significantly enhanced TrkA phosphorylation in response to brief NGF stimulation. Two downstream signaling pathways (Erk1/2 and Akt) that are directly activated in response to phospho-TrkA were differentially affected. Akt signaling, arising in response to activated TrkA at the plasma membrane was unaffected. However Erk1/2 phosphorylation, triggered on signaling endosomes, was increased. Cytoplasmic phospho-Erk1/2 persisted at elevated levels relative to control samples for up to 24 h following NGF stimulation. Nuclear shuttling of phospho Erk1/2, which is required to induce MAPK phosphatase expression and down regulate signaling, was greatly reduced by the Rab7 CMT2B mutants and explains the previously reported inhibition in PC12 neurite outgrowth. In conclusion, the data demonstrate a mechanistic link between Rab7 CMT2B mutants and altered TrkA and Erk1/2 signaling from endosomes.     

p53 is required for nerve growth factor-mediated differentiation of PC12 cells via regulation of TrkA levels

p53 is necessary for the elimination of neural cells inappropriately differentiated or in response to stimuli. However, the role of p53 in neuronal differentiation is not certain. Here, we showed that nerve growth factor (NGF)-mediated differentiation in PC12 cells is enhanced by overexpression of wild-type p53 but inhibited by mutant p53 or knockdown of endogenous wild-type p53, the latter of which can be rescued by expression of exogenous wild-type p53. Interestingly, p53 knockdown or overexpression of mutant p53 attenuates NGF-mediated activation of TrkA, the high-affinity receptor for NGF and a tyrosine kinase, and activation of the mitogen-activated protein kinase pathway. In addition, p53 knockdown reduces the constitutive levels of TrkA, which renders PC12 cells inert to NGF. And finally, we showed that both constitutive and stimuli-induced expressions of TrkA are regulated by p53 and that induction of TrkA by activated endogenous p53 enhances NGF-mediated differentiation. Taken together, our data demonstrate that p53 plays a critical role in NGF-mediated neuronal differentiation in PC12 cells at least in part via regulation of TrkA levels.     

Separate Cyclic AMP Sensors for Neuritogenesis,Growth Arrest,and Survival of Neuroendocrine Cells

Dividing neuroendocrine cells differentiate into a neuronal-like phenotype in response to ligands activating G protein-coupled receptors, leading to the elevation of the second messenger cAMP. Growth factors that act at receptor tyrosine kinases, such as nerve growth factor, also cause differentiation. We report here that two aspects of cAMP-induced differentiation, neurite extension and growth arrest, are dissociable at the level of the sensors conveying the cAMP signal in PC12 and NS-1 cells. Following cAMP elevation, neuritogenic cyclic AMP sensor/Rapgef2 is activated for signaling to ERK to mediate neuritogenesis, whereas Epac2 is activated for signaling to the MAP kinase p38 to mediate growth arrest. Neither action of cAMP requires transactivation of TrkA, the receptor for NGF. In fact, the differentiating effects of NGF do not require activation of any of the cAMP sensors protein kinase A, Epac, or neuritogenic cyclic AMP sensor/Rapgef2 but, rather, depend on ERK and p38 activation via completely independent signaling pathways. Hence, cAMP- and NGF-dependent signaling for differentiation are also completely insulated from each other. Cyclic AMP and NGF also protect NS-1 cells from serum withdrawal-induced cell death, again by two wholly separate signaling mechanisms, PKA-dependent for cAMP and PKA-independent for NGF.     

Cyclooxygenase 2 promotes cell survival by stimulation of dynein light chain expression and inhibition of neuronal nitric oxide synthase activity

Cyclooxygenase 2 (COX-2) inhibits nerve growth factor (NGF) withdrawal apoptosis in differentiated PC12 cells. The inhibition of apoptosis by COX-2 was concomitant with prevention of caspase 3 activation. To understand how COX-2 prevents apoptosis, we used cDNA expression arrays to determine whether COX-2 regulates differential expression of apoptosis-related genes. The expression of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase [PIN]) was significantly stimulated in PC12 cells overexpressing COX-2. The COX-2-dependent stimulation of DLC expression was, at least in part, mediated by prostaglandin E(2). Overexpression of DLC also inhibited NGF withdrawal apoptosis in differentiated PC12 cells. Stimulation of DLC expression resulted in an increased association of DLC/PIN with neuronal nitric oxide synthase (nNOS), thereby reducing nNOS activity. Furthermore, nNOS expression and activity were significantly increased in differentiated PC12 cells after NGF withdrawal. This increased nNOS activity as well as increased nNOS dimer after NGF withdrawal were inhibited by COX-2 or DLC/PIN overexpression. An nNOS inhibitor or a membrane-permeable superoxide dismutase (SOD) mimetic protected differentiated PC12 cells from NGF withdrawal apoptosis. In contrast, NO donors induced apoptosis in differentiated PC12 cells and potentiated apoptosis induced by NGF withdrawal. The protective effects of COX-2 on apoptosis induced by NGF withdrawal were also overcome by NO donors. These findings suggest that COX-2 promotes cell survival by a mechanism linking increased expression of prosurvival genes coupled to inhibition of NO- and superoxide-mediated apoptosis.     

Multiple Pathways of N-Kinase Activation in PC12 Cells

Past work established a cell-free assay for a nerve growth factor (NGF)-activated protein kinase activity (designated N-kinase) that utilizes tyrosine hydroxylase and histone H1 as substrates and that is distinct from a variety of well-characterized kinases. This study explores the specificity and mechanistic pathway(s) by which N-kinase activity is regulated in PC12 rat pheochromocytoma cells. N-kinase is rapidly activated in these cells by treatment with NGF, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), phorbol ester, or dibutyryl cyclic AMP. Our data indicate that the stimulated activity is the same for each agent by several criteria: It exhibits the same characteristic biphasic elution pattern by Mono S fast protein liquid chromatography (FPLC), except for the case of dibutyryl cyclic AMP in which one of the activity peaks is somewhat shifted; it shows the same elution pattern by FPLC on a Superose 12 column; it possesses identical substrate specificity; and, except in the case of dibutyryl cyclic AMP, it does not show additivity when each agent is added simultaneously with NGF. The multiple forms of N-kinase are interconvertible in that rechromatography on a Mono S column yields a single peak of activity. Also, when NGF and dibutyryl cyclic AMP are simultaneously presented to cells, the chromatographic profile resembles that with NGF alone. Activation occurs through several independent initial pathways. Down-regulation of protein kinase C by phorbol ester pretreatment prevents N-kinase activation by phorbol ester, but not by the other agents. A PC12 cell-derived line deficient in cyclic AMP-dependent protein kinase II activity exhibits N-kinase activation by all treatments except dibutyryl cyclic AMP. The properties of N-kinase suggests that it is similar or identical to the ribosomal S6 protein kinase described by Blenis and Erikson. Additional experiments revealed that N-kinase activity can be stimulated in several cell lines in addition to PC12 cells. These findings indicate that the N-kinase can be activated via multiple second-messenger pathways and that it could therefore potentially play a significant role in mediating shared intracellular responses to various extracellular signals.     

Ras-Regulated Hypophosphorylation of the Retinoblastoma Protein Mediates Neuronal Differentiation in PC12 Cells

Abstract: To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12- O -tetradecanoylphorbol 13-acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin-6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa-Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras-dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells.     

Nerve Growth Factor (NGF) Induces a Rapid and Sustained Downregulation of the Focal Adhesion Kinase (FAK)

1. Exposure of PC12 cells to nerve growth factor (NGF) induces an early tyrosine phosphorylation of many proteins, a number of which is still unidentified. Although NGF is known to bind to and activate the receptor tyrosine kinase TrkA, many downstream targets of NGF signaling may be possibly phosphorylated by nonreceptor tyrosine kinases such as c-Src and focal adhesion kinase (FAK). 2. In the present study, exposure of TrkA-overexpressing PC12 cells to NGF is found to cause a rapid and sustained loss in the recovery of a subpopulation of nominally active FAK (i.e., being autophosphorylated on the positive site of regulation). 3. Consistent with the possibility that NGF induces the proteolysis of FAK via recruitment of Src family kinases, the use of various phosphorylation site-specific anti-FAK antibodies revealed an NGF-inducible and PP1-sensitive accumulation of a putative fragment (i.e., p62) of FAK. Significantly, the mitogenic epidermal growth factor (EGF) failed to induce the downregulation of FAK and the accumulation of tyrosine phosphorylated p62. Such differential response of FAK to NGF and EGF may shape the specificity by which these growth factors control the status of cell-matrix adhesion and the adhesion-driven signaling.     

SH2-B is required for nerve growth factor-induced neuronal differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced neuronal differentiation.     

Cholera Toxin and Dibutyryl Cyclic AMP Inhibit the Expression of Neurofilament Protein Induced by Nerve Growth Factor in Cultures of Naive and Primed PC12 Cells

The effects of nerve growth factor (NGF), dibutyryl cyclic AMP (db cAMP), and cholera toxin on neurofilament protein expression in cultures of PC12 rat pheochromocytoma cells were examined using an enzyme-linked immunoadsorbent assay (ELISA). Morphological differentiation induced by NGF was associated with up to 30-fold increases in the level of neurofilament protein recognised by monoclonal antibody RT97. A more rapid response was apparent from primed as compared to naive PC12 cells. Cholera toxin and db cAMP both induced morphological differentiation of naive PC12 cells, but failed to promote neurite regeneration from primed cells. Neither response was associated with a significant induction of neurofilament protein. Both cholera toxin and db cAMP, but not B-cholera toxin nor antibodies to the toxin receptor, were found to inhibit the neurofilament protein response induced by NGF. Primed cells were more susceptible to this inhibition, and both cholera toxin and db cAMP inhibited neurite regeneration from these cells. These data suggest that increased intracellular cyclic AMP can suppress the expression of neuronal differentiation antigens induced by NGF, and are consistent with a role for neurofilament protein in promoting or facilitating the formation of a stable neuritic network.     

Nerve growth factor specifically stimulates translation of eukaryotic elongation factor 1A-1 (eEF1A-1) mRNA by recruitment to polyribosomes in PC12 cells

During postnatal brain development the level of peptide elongation factor-1A (eEF1A-1) expression declines and that of the highly homologous isoform, eEF1A-2, increases in neurons. eEF1A-1 is implicated in cytoskeletal interactions, tumorigenesis, differentiation, and the absence of eEF1A-2 is implicated in neurodegeneration in the mouse mutant, wasted. The translation of eEF1A-1 mRNA is up-regulated via mitogenic stimulation. However, it is not known if eEF1A-1 mRNA translation is regulated by neurotrophins or if its synthesis is differentially regulated than that of the neuronal isoform, eEF1A-2. Regulated translation of these factors by neurotrophins, particularly by the Trk class of neurotrophin receptors, would implicate them in differentiation, survival, and neuronal plasticity. In this study, we investigated the effect of nerve growth factor (NGF) stimulation on the synthesis of eEF1A-1 and eEF1A-2. We found that NGF stimulation causes a preferential synthesis of eEF1A-1 over eEF1A-2 in PC12 cells. We analyzed the co-sedimentation of eEF1A-1 mRNA with polyribosome fractions in sucrose gradients, and found that NGF stimulation enriched the presence of eEF1A-1 mRNA in polyribosomes, indicating that the translation of eEF1A-1 mRNA is regulated by NGF. Inhibitors of phosphatidylinositol 3-kinase (LY 294002), mammalian target of rapamycin (rapamycin), and the NGF receptor, TrkA (K-252a), but not of mitogen-activated protein kinase (PD 98059), prevented the recruitment of eEF1A-1 mRNA to polyribosomes. The mobilization of eEF1A-1 mRNA to polyribosomes was rapamycin-sensitive in both proliferating and differentiated PC12 cells, indicating the importance of this pathway during differentiation. Our data shows that after growth factor withdrawal, an NGF-signaling pathway stimulates eEF1A-1 mRNA translation in proliferating and differentiated PC12 cells. Therefore, eEF1A-1 mRNA is a specific translational target of TrkA signaling.     

The nerve growth factor precursor proNGF exhibits neurotrophic activity but is less active than mature nerve growth factor

Nerve growth factor (NGF) promotes neuronal survival and differentiation and stimulates neurite outgrowth. NGF is synthesized as a precursor, proNGF, which undergoes post-translational processing to generate mature beta-NGF. It has been assumed that, in vivo, NGF is largely processed into the mature form and that mature NGF accounts for the biological activity. However, we recently showed that proNGF is abundant in CNS tissues whereas mature NGF is undetectable, suggesting that proNGF has biological functions beyond its role as a precursor. To determine whether proNGF exhibits biological activity, we mutagenized the precursor-processing site and expressed unprocessed, cleavage-resistant proNGF protein in insect cells. Survival and neurite outgrowth assays on murine superior cervical ganglion neurons and PC12 cells indicated that proNGF exhibits neurotrophic activity similar to mature 2.5S NGF, but is approximately fivefold less active. ProNGF binds to the high-affinity receptor, TrkA, as determined by cross-linking to PC12 cells, and is also slightly less active than mature NGF in promoting phosphorylation of TrkA and its downstream signaling effectors, Erk1/2, in PC12 and NIH3T3-TrkA cells. These data, coupled with our previous report that proNGF is the major form of NGF in the CNS, suggest that proNGF could be responsible for much of the biological activity normally attributed to mature NGF in vivo.     

Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.     

The Csk homologous kinase associates with TrkA receptors and is involved in neurite outgrowth of PC12 cells.

Csk homologous kinase (CHK), a member of the Csk regulatory tyrosine kinase family, is expressed primarily in brain and hematopoietic cells. The role of CHK in the nervous system is as yet unknown. Using PC12 cells as a model system of neuronal cells, we show that CHK participates in signaling mediated by TrkA receptors. CHK was found to be associated with tyrosine-phosphorylated TrkA receptors in PC12 cells upon stimulation with NGF. Binding assays and far Western blotting analysis, using glutathione S-transferase fusion proteins containing the Src homology 2 (SH2) and SH3 domains of CHK, demonstrate that the SH2 domain of CHK binds directly to the tyrosine-phosphorylated TrkA receptors. Site-directed mutagenesis of TrkA cDNA, as well as phosphopeptide inhibition of the in vitro interaction of the CHK-SH2 domain or native CHK with TrkA receptors, indicated that the residue Tyr-785 on TrkA is required for its binding to the CHK-SH2 domain upon NGF stimulation. In addition, overexpression of CHK resulted in enhanced activation of the mitogen-activated protein kinase pathway upon NGF stimulation, and microinjection of anti-CHK antibodies, but not anti-Csk antibodies, inhibited neurite outgrowth of PC12 cells in response to NGF. Thus, CHK is a novel signaling molecule that participates in TrkA signaling, associates directly with TrkA receptors upon NGF stimulation, and is involved in neurite outgrowth of PC12 cells in response to NGF.     

A Nerve Growth Factor-Dependent Protein Kinase That Phosphorylates Microtubule-Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF-pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three- to eightfold in terms of phosphorylation of microtubule-associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP(dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP-dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF; it required Mg²⁺ for activity but not Mn²⁺ or Ca²⁺. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF-dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K-252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF-dependent MAP kinase was selectively abolished. We isolated several mutant clones of PC12D cells that were deficient in the ability to induce neurites in response to either of the two stimulators. In these variant cells, the activity of the relevant protein kinase was decreased, in parallel with the deficiency in the neurite response to NGF or dbcAMP. These observations suggest that the NGF-dependent MAP kinase may play an important role in the outgrowth of neurites from PC12 cells in response to NGF.     

Neuronal outgrowth and rescue induced by cyclic phosphates in PC12 cells.

A series of cyclic glycerophosphates and their deoxy analogues were tested for induction of neuronal outgrowth in PC12 cells. Under chronic presence of a cyclic phosphate PC12 cells developed distinct isles of neuronal networks which covered up to 20% of the culture area, while alpha and beta glycerophosphates (the negative control compounds) did not induce any neuronal outgrowth. Distinct isles of neuronal networks were also observed upon short term application (i.e. 2 pulses of 3 hours each at day 1 and day 4) of the tested cyclic phosphates in contrast to an analogous short term exposure to NGF which was abortive. Analysis of tyrosine phosphorylation indicated a battery of phosphorylated proteins after several minutes of application of the cyclic phosphates, among which was an ERK protein of approximately 63 kD (possibly ERK7). Nerve rescue experiments were carried out with NGF differentiated PC12 cells where NGF was replaced with either 1,2 or 1,3 cyclic propanediolphosphate (1,2 cPP and 1,3 cPP) for 7 days. A distinct dose dependent preservation of neuronal network by these compounds was observed. In the control cultures NGF deprivation resulted in massive neuronal retraction and cell death. Preliminary experiments indicated that the nerve rescue by the cyclic phosphates involves the increase in the level of CASPase 6. The above findings suggest that cyclic glycerophosphates and their analogues may bear important physiological and pharmacological implications which are currently under investigation.