trkC Transcripts Research Articles

Experimental verification of a predicted novel microRNA located in human PIK3CA gene with a potential oncogenic function in colorectal cancer.

PI3K/AKT signaling is involved in cell survival, proliferation, and migration. In this pathway, PI3Kα enzyme is composed of a regulatory protein encoded by p85 gene and a catalytic protein encoded by PIK3CA gene. Human PIK3CA locus is amplified in several cancers including lung and colorectal cancer (CRC). Therefore, microRNAs (miRNAs) that are encoded within the PIK3CA gene might have a role in cancer development. Here, we report a novel microRNA named PIK3CA-miR1 (EBI accession no. LN626315), which is located within PIK3CA gene. A DNA segment corresponding to PIK3CA-premir1 sequence was transfected in human cell lines that resulted in generation of mature exogenous PIK3CA-miR1. Following the overexpression of PIK3CA-miR1, its predicted target genes (APPL1 and TrkC) were significantly downregulated in the CRC-originated HCT116 and SW480 cell lines, detected by qRT-PCR. Then, dual luciferase assay supported the interaction of PIK3CA-miR1 with APPL1 and TrkC transcripts. Endogenous PIK3CA-miR1 expression was also detected in several cell lines (highly in HCT116 and SW480) and highly in CRC specimens. Consistently, overexpression of PIK3CA-premir1 in HCT116 and SW480 cells resulted in significant reduction of the sub-G1 cell distribution and apoptotic cell rate, as detected by flowcytometry, and resulted in increased cell proliferation, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PIK3CA-miR1 overexpression also resulted in Wnt signaling upregulation detected by Top/Fop assay. Overall, accumulative evidences indicated the presence of a bona fide novel onco-miRNA encoded within the PIK3CA oncogene, which is highly expressed in colorectal cancer and has a survival effect in CRC-originated cells.

Early neuronal progenitor cell line expressing solely non-catalytic isoform of TrkC

TrkC is a receptor for neurotrophin-3 that regulates development of neuronal precursors. Transduction of signals into receptor-dependent signaling pathways is mainly due to the activation of the intrinsic tyrosine kinase of the TrkC receptor. Alternative splicing of the trkC transcripts generates catalytic and non-catalytic isoforms. The non-catalytic isoform, denoted as TrkC-NC2, contains unique sequence, instead of deleted entire kinase domain. Here, we report that neural cell line MB-G, derived from brain of embryos of transgenic tsA58-SV40 mice, contains mRNA encoding TrkC-NC2 without concomitant expression of mRNA for catalytic TrkC molecule.

Gene expression of TrkC (NTRK3) in human soft tissue tumours.

TrkC is a member of the Trk family of tyrosine kinase receptors and plays an important role in the development and maintenance of neural tissues. Although a variety of non-neuronal tissues have also been shown to express TrkC, the status of TrkC in soft tissue tumours has been poorly investigated, except for a small fraction of tumours including congenital/infantile fibrosarcoma characterized by an ETV6-NTRK3 (also known as Tel-TrkC) fusion gene. To broaden knowledge about the TrkC status in human neoplasms, the expression of TrkC transcripts was assessed in 51 soft tissue tumours of variable lines of differentiation by reverse transcription-polymerase chain reaction (RT-PCR), using primer sets flanking their extracellular domain, the tyrosine kinase domain, and the intracellular domain of a truncated variant (Trunc 1) described previously. In 44 of the 51 tumours, TrkC transcripts, including alternatively spliced isoforms, were detected. The truncated transcripts (Trunc 1) were co-expressed in 40 of the 44 tumours and were expressed in one tumour without native TrkC gene expression. In two of the remaining six tumours, part of the sequence coding the tyrosine kinase domain of TrkC appeared to be truncated. Using a 3' rapid amplification of cDNA ends (3'RACE) method, another truncated isoform (Trunc 2) was isolated from one of the tumours, in which the TrkC transcript was terminated with a novel 160-base pair sequence. This truncated isoform was identified in nine of the 51 tumours examined by RT-PCR using primers for Trunc 2. There was no clear correlation between the types of TrkC isoforms detected and histological types or grades of the tumours. These results suggest that human soft tissue tumours widely express TrkC, irrespective of their cellular lineage, morphology, and biological behaviour. Dysregulated TrkC expression may enhance overgrowth or transformation of various mesenchymal cells.

Selective Regulation of trkC Expression by NT3 in the Developing Peripheral Nervous System

We have studied the influence of neurotrophin-3 (NT3) on the expression of its receptor tyrosine kinase, trkC, in embryonic mice. The expression of trkC transcripts encoding full-length and kinase-deficient receptors was almost entirely restricted to neurons in the trigeminal ganglion and increased markedly throughout development. In NT3(+/-) embryos, the level of trkC mRNA in the trigeminal ganglion was much lower than that in wild-type embryos, although there was no significant reduction in the total number of neurons in the ganglion. This demonstrates that endogenous NT3 regulates trkC expression in trigeminal neurons independently of changes in population size. In NT3(-/-) embryos, the number of neurons in the trigeminal ganglion was much lower than in wild-type embryos, and there was a further reduction in the mean neuronal level of trkC mRNA. Direct regulation of trkC mRNA expression in cultured trigeminal neurons was also observed, although the finding that trkC mRNA levels were sustained better in explant cultures than in dissociated cultures irrespective of the presence of NT3 suggests that trkC mRNA expression is regulated by additional factors within the ganglion. In contrast to trigeminal neurons, the level of trkC mRNA was sustained at normal levels in neurons of the sympathetic chain of NT3(-/-) embryos and was not increased by NT3 in sympathetic neuron cultures. TrkC mRNA expression in developing cutaneous tissues was also unaffected by the NT3 null mutation. In summary, our findings provide the first clear evidence that the expression of a trk receptor, tyrosine kinase, is regulated by physiological levels of its ligand in vivo and show that regulation by NT3 is cell type-specific.

Neurotrophin receptors in the geniculate ganglion

We examined the distribution of the high affinity neurotrophin receptors (trkA, trkB, and trkC) in the rat geniculate ganglion. Previous work had shown that during early (prenatal) development, trkB and its two ligands, BDNF and NT-4/5, were most important for survival of almost all neurons. Using nested polymerase chain reaction (PCR), we showed that trkA, trkB, and trkC transcripts were expressed, and the mRNAs for trkB and trkC were more abundant than that for trkA. We modified and improved the method for direct reverse transcription in situ PCR and localized trkB mRNA in approximately one third of the neurons in the ganglion. Immunohistochemical data confirmed that approximately the same fraction of neurons was immunoreactive with antibody vs. trkB, and an approximately equal fraction was immunoreactive with trkC antibody. These results are consistent with the notion that both BDNF/trkB and NT-3/trkC play important roles in maintenance of the geniculate ganglion neurons and possibly the peripheral taste system in the young postnatal rat.

Development of striatal dopaminergic function. III: Pre- and postnatal development of striatal and cortical mRNAs for the neurotrophin receptors trkBTK+ and trkC and their regulation by synaptic dopamine.

Known neurotrophins in the nigrostriatal system include brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) which exert biological effects after high affinity binding to their respective tyrosine kinase receptors, trkB and trkC. We measured striatal trkBTK+ and trkC mRNAs in rat brain sections with in situ hybridization histochemistry. Both trkBTK+ and trkC transcripts were present in the striatal anlage at embryonic day (E) 14 or 16. Striatal trkBTK+ mRNA levels increased to a peak in the late prenatal period and gradually declined in the postnatal period. In contrast, striatal trkC mRNA levels peaked on E16, then declined to fairly constant levels. Striatal trkBTK+ gene expression increased from the medial to lateral quadrants throughout development, whereas trkC mRNA increased from the lateral to medial quadrants prenatally but increased from the dorsal to ventral quadrants on postnatal days (P) 1 and 3. The distinct spatiotemporal developmental profiles of trkBTK+ and trkC mRNA suggest that their respective ligands BDNF and NT-4/5, and NT-3, may have specialized functions in striatal neuronal development. Because neurotransmitters may regulate neurotrophin function in developing neural systems, we treated rats of various ages with the indirect dopamine agonist cocaine and measured the effects of cocaine treatment on transcription of the trk genes. Acute 1 h cocaine treatment increased trkBTK+ and trkC mRNA levels in the P5 striatum but not in the E15, E20, or adult striatum. The trkBTK+ effect was blocked by pretreatment with the D1 receptor antagonist, SCH23390, and was not affected by pretreatment with the D2 receptor antagonist, eticlopride. In contrast, trkC regulation may be mediated by independent stimulation of D1 and D2 receptors. We hypothesize that the endogenous nigrostriatal neurotransmitter dopamine can modulate striatal neurotrophin responsiveness and thereby influence striatal neuronal development during a defined developmental period.

Development of striatal dopaminergic function. III: Pre- and postnatal development of striatal and cortical mRNAs for the neurotrophin receptors trkB TK+ and trkC and their regulation by synaptic dopamine

Known neutrophins in the nigrostriatal system include brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) which exert biological effects after high affinity binding to their respective tyrosine kinase receptors, trkB and trkC. We measured striatal trkB TK+ and trkC mRNAs in rat brain sections with in situ hybridization histochemistry. Both trkB TK+ and trkC transcripts were present in the striatal anlage at embryonic day (E) 14 or 16. Striatal trkB TK+ mRNA levels increased to a peak in the late prenatal period and gradually declined in the postnatal period. In contrast, striatal trkC mRNA levels peaked on E16, then declined to fairly constant levels. Striatal trkB TK+ gene expression increased from the medial to lateral quadrants throughout development, whereas trkC mRNA increased from the lateral to medial quadrants prenatally but increased from the dorsal to ventral quadrants on postnatal days (P) 1 and 3. The distinct spatiotemporal developmental profiles of trkB TK+ and trkC mRNA suggest that their respective ligands BDNF and NT-4/5, and NT-3, may have specialized functions in striatal neuronal development. Because neurotransmitters may regulate neurotrophin function in developing neural systems, we treated rats of various ages with the indirect dopamine agonist cocaine and measured the effects of cocaine treatment on transcription of the trk genes. Acute 1 h cocaine treatment increased trkB TK+ and trkC mRNA levels in the P5 striatum but not in the E15, E20, or adult striatum. The trkB TK+ effect was blocked by pretreatment with the D1 receptor antagonist, SCH23390, and was not affected by pretreatment with the D2 receptor antagonist, eticlopride. In contrast, trkC regulation may be mediated by independent stimulation of D1 and D2 receptors. We hypothesize that the endogenous nigrostriatal neurotransmitter dopamine can modulate striatal neurotrophin responsiveness and thereby influence striatal neuronal development during a defined developmental period.

Neurotrophin activates signal transduction in oligodendroglial cells: expression of functional TrkC receptor isoforms.

The role of the NT-3 has been implicated in the survival of progenitor oligodendrocytes in culture. The object of this study was to investigate the expression of the TrkC receptor and its responsiveness in glial cells. We report the expression of two TrkC receptor isoforms in rat primary oligodendrocyte cultures, a glial progenitor cell line, CG-4, and in C6 glioma cells. The reverse transcription-polymerase chain reaction-aided amplification of glial trkC with specific primers from the kinase domain, followed by its cloning and sequencing, shows the presence of two trkC transcripts. The sequence of one of the transcripts is homologous to a previously identified trkC isoform which encodes a functional receptor. The other transcript contains a 42-bp insert in the kinase domain. A Western blot of CG-4 and C6 probed with antibody to a TrkC revealed the presence of gp145-kDa protein band. The investigations revealed a rapid autophosphorylation of gp145TrkC in CG-4 and C6 cells in the presence of its specific ligand, NT-3. Furthermore, K252a, a neurotrophin-specific inhibitor, abolishes the NT-3-mediated receptor autophosphorylation. We also examined other NT-3-dependent phosphorylation of cellular substrates in oligodendroglial cells. Interestingly, we observed phosphorylation of phospholipase C gamma-1 in CG-4 and C6 cells, and phosphorylation of phosphatidylinositol 3-kinase in C6 cells in the presence of NT-3. Both the NT-mediated phosphorylation of phospholipase C gamma-1 and phosphorylation of phosphatidylinositol 3-kinase are blocked in the presence of K252a. The detection of the NT-3-mediated early signal transduction events demonstrates that TrkC receptor exhibits NT-3-mediated intracellular response in oligodendroglial cells.

Neurotoxic injury in rat hippocampus differentially affects multiple trkB and trkC transcripts

In the present work we determined, by Northern blotting, ribonuclease assay and in situ hybridization, the level of multiple trkB and trkC transcripts at different times after ibotenic acid-induced neuronal injury in the rat hippocampus. All the transcripts (7.0–7.5, 2.4 and 1.8 kb) encoding the truncated TrkB receptor are coordinately up-regulated following neurotoxic injury, with a time-course similar to that observed for the glial fibrillary acidic protein tnRNA, a molecular marker of reactive astrocytes. The highest level of induction was observed for the 2.4 kb mRNA level. The 1.8 kb mRNA, whose relative level is higher in astroglial cultures compared to normal brain tissue, is detectable only in the gliotic hippocampus. The 9 kb trkB rnRNA, which encodes the full-length TrkB receptor, rapidly decreases with a time-course similar to that previously observed for other neuronal markers. In situ hybridization studies show that the increased mRNA level per cell is a major determinant in the up-regulation of truncated trkB expression. A decrease of truncated and full-length trkC mRNA was observed in the neuron-depleted astroglia-enriched hippocampus, suggesting that this mRNA is mainly localized in the neuronal layers and that no induction of its expression occurs in reactive astrocytes.

Developmental regulation of full-length trkC in the rat sciatic nerve.

In order to gain insight into potential roles of neurotrophins in Schwann cell biology, the expression of neurotrophin receptors of the trk gene family was investigated in rat sciatic nerve development. This analysis revealed differential regulation of truncated and full-length receptors. TrkA was undetectable even when analysed with a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) method. TrkB was present at the mRNA as well as protein level only in its truncated form. Surprisingly, multiple isoforms of trkC, including full-length forms, were detected in early postnatal nerve. Specific antibodies detected truncated and full-length trkC proteins in Western blotting, and RT-PCR revealed the presence of two full-length isoforms, one of them containing the 14 amino acid kinase insert. In situ hybridisation localized the expression of trkC to a subpopulation of Schwann cells. TrkC receptors are expressed already in nerves from day-16 embryos. In contrast to early postnatal stages, full-length trkC receptors are no longer expressed in adult nerves, which, however, maintain expression of truncated trkC transcripts. The presence of trkC kinases in peripheral nerve suggests a role for neurotrophin-3, the only known trkC ligand, in peripheral nerve development.

Muscle sensory neurons require neurotrophin-3 from peripheral tissues during the period of normal cell death

To determine if muscle sensory neurons require neurotrophin-3 (NT3) during the period of normal cell death, we used an NT3-specific antiserum to deplete NT3 from peripheral tissues during this period in chick embryos. DiI staining of dorsal roots indicated that limb injections of anti-NT3 reduced the spinal projection of muscle spindle afferents. In contrast, injection of the antiserum into the spinal cord had no demonstrable effect, indicating that the reduced projection following limb injection was due to peripheral blockade of NT3 signaling. Counts of neurons retrogradely labeled from muscle and cutaneous nerves showed that peripheral blockade of NT3 selectively reduced the survival of muscle sensory neurons without affecting the survival of cutaneous sensory neurons or motoneurons. In situ hybridization with trkC probes indicated that, during the period of cell death, most large diameter muscle sensory neurons express trkC transcripts, whereas few cutaneous neurons express this receptor for NT3. We conclude that large diameter muscle afferents, including spindle afferents, require NT3 from peripheral tissues to survive the normal period of sensory neuron death in vivo.

Isoforms of the avian TrkC receptor: A novel kinase insertion dissociates transformation and process outgrowth from survival

TrkC receptor isoforms have been identified by cDNA cloning and RT-PCR analysis of embryonic chick brain RNA. An N-terminal truncation motif is missing from the signal sequence and first cysteine cluster of the extracellular domain. Within the cytoplasmic dimain, a kinase truncation motif retains part of the kinase domain, but appeared to lack activity. Finally, a kinase insert (KI) motif introduces a 25 amino acid sequence distinct from the known mammalian inserts. KI receptors, like full-length receptors, were tyrosine phosphorylated in response to NT-3 and mediated the transformation of chick embryo fibroblasts and process outgrowth from rat PC12 cells. However, KI receptors supported little, if any, survival of serum-deprived PC12 cells. These results indicate that alternative splicing of trkC transcripts is an important mechanism for regulating cellular responses to NT-3.

Expression of polyadenylated and non-polyadenylated TrkC transcripts in the roent central nervous system

We have previously isolated a full-length cDNA clone encoding rat TrkC, a member of the Trk family of tyrosine kinase receptors, that specifically mediates biological responses to neurotrophin-3. Here, we report the identification of five major trkC transcripts in the adult and developing rat and mouse brain suggesting the presence of several TrkC receptors. Northern blot hybridizations revealed that three of these trk C transcripts (of 14, 3.9 and 4.8 kb) were poly(A) + messenger RNAs, while the two others, of shorter size (1.1 and 0.7 kb), were poly(A) − messenger RNAs. All transcripts were expressed in 11 brain regions but poly(A) − messenger RNAs were found at much higher levels than poly(A) + messenger RNAs in the cerebellum. Hybridization with five oligonucleotide and two complementary DNA probes, corresponding to different parts of the full-length trkC complementary DNA, revealed that the two poly(A) − transcripts may encode for receptors truncated in their extracellular and kinase intracellular domains. During ontogeny, poly(A) − trkC messenger RNAs were found at low amounts at prenatal and early postnatal ages with a drastic increase in the cerebellum at postnatal day 30. No poly(A) − transcript was identified for the trk B gene. In situ hybridization revealed that trkC messenger RNAs are expressed both in granular and Purkinje cells in the cerebellum. Northern blot on RNA extracted from mice mutant strains with degeneration of either granular or both granular and Purkinje cells suggested that poly(A) − and poly(A) + trkC messenger RNAs are expressed within the same cells. We have demonstrated the existence of several trkC transcripts that differ both by their size and polyadenylation. This phenomenon could be of physiological relevance in regulating TrkC functions. To the best of our knowledge, this is an original feature for a mammalian gene expression. Studies focused on poly(A) − messenger RNAs could give rise to the identification of other genes expressed in a similar fashion.

Developmental expression of trkC, the neurotrophin-3 receptor, in the mammalian nervous system

The mammalian trkC gene encodes a tyrosine protein kinase that serves as a functional receptor for neurotrophin-3 (NT-3). Here, we report that trkC is widely expressed in the developing and adult nervous system. Using in situ hybridization, we first detect trkC transcripts in the telencephalon and spinal cord of embryonic day 9.5 mouse embryos. In later embryonic development, trkC is expressed in various structures of the CNS including the caudatoputamen, septal nuclei, cerebellum, and brainstem. In the PNS, trkC hybridization appears to correlate, both temporally and spatially, with the outgrowth of axons toward their peripheral targets. trkC transcripts were also identified in the autonomous enteric nervous system as well as in some non-neural tissues such as the wall of the aorta and the acini of the submaxillary and sublingual glands. In the adult mouse, trkC gene expression is heterogeneously distributed throughout the brain, with highest levels in limbic and diencephalic structures. These results indicate that the trkC gene is widely expressed in the three identified branches of the mammalian nervous system and appears to correlate with the expression of NT-3, its cognate ligand. The apparent colocalization of trkC transcripts with NT-3 raises the possibility this neurotrophin exerts its trophic effects by a paracrine and/or autocrine mechanism.

Expression of mRNAs for neurotrophin receptors in the dorsal root ganglion and spinal cord during development and following peripheral or central axotomy

Expression of mRNAs for the protein tyrosine kinases trk, trkB and trkC, encoding essential components of high-affinity neurotrophin receptors, was studied in the spinal cord and dorsal root ganglion during normal development and in the adult rat following peripheral and central axon injury. Northern blots revealed multiple trkB transcripts in the embryonic, early postnatal and adult spinal cord with different patterns of expression during development. The levels of 9.0 kb and 4.8 trkB transcripts, encoding a full-length trkB receptor, increased progressively during embryonic development with maximal levels around birth, followed by a decline at adulthood. In contrast, the level of 7.5 7.0 kb trkB transcripts, encoding a truncated trkB receptor, reached maximal levels shortly after birht adn similar levels remained in the adult animal. In the spinal cord a 4.7kb trkC transcript was detected with maximal levels shortly after birth. In situ hybridization revealed a uniform labeling throughout the spinal cord for both trkB and trkC mRNAs with maximal intensities of labeling shortly after birth. The level of the 2.4 kb trkB transcript in the spinal cord increased 5-fold 8 days after a crush lesion of the sciatic nerve or the dorsal root, while no change was seen in the levels of the other trkB transcripts. No change in the 4.7 kb trkC mRNA was seen following these two injuries, although increased levels of several smaller size trkC transcripts were observed. For both trkB and trkC, similar size transcripts as seen in the spinal cord were also detected in adult rat dorsal root ganglia. Consistent with previous observations of decreased levels of cytoskeletal proteins after peripheral and central axotomy, the level of neurofilment light chain mRNA decreased markedly in the dorsal root ganglia following a crush lesion of the sciatic nerve or of the dorsal root. A small decrease was also seen in the level of preprotachykinin-A mRNA encoding the protein precursor of substance P. In the same animals, the levels of all five trkB transcripts increased 3-fold in the dorsal root ganglia in response to these two injuries. A small increase was also seen in the level of trkC mRNA. The level of brain-derived neurotrophic factor (BDNF) mRNA increased two-fold in the dorsal root ganglia following either of the two lesions, while no change was detected in trk mRNA following these two injuries. These data show that expression of the members of the trk family of protein tyrosine kinase receptors is differentially regulated during spinal cord development and that they exhibit different lesion responses to axotomy. The differential expression of transcripts encoding full-length or truncated trkB receptors in the developing and injured spinal cord implies different functional roles for these two receptor subtypes. The functional consequences of these changes are discussed in view of the current knowledge of the action of neurotrophins.

TrkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3

We report the isolation and molecular characterization of trkC a new member of the trk family of tyrosine protein kinase genes. trkC is preferentially expressed in the brain. In situ hybridization studies revealed trkC transcripts in the hippocampus, cerebral cortex, and the granular cell layer of the cerebellum. The product of the trkC gene has been identified as a glycoprotein of 145,000 daltons, gp145 trkC which is equally related to the previously characterized gp140 trk and gp145 trkB tyrosine kinases. gp145 trkC is a functional receptor for neurotrophin-3 (NT-3). However, gp145 trkC does not bind the highly related neurotrophic factors NGF or BDNF. In proliferating cells, the interaction between gp145 trkC and NT-3 elicits a more efficient biological response than when NT-3 binds to its other receptors gp140 trk and gp145 trkB These results indicate that gp145 trkC may play an important role in mediating the neurotrophic effects of NT-3.