Expression Of LAR Research Articles

Metabolome integrated with transcriptome reveals the mechanism of three different color formations in Taxus mairei arils.

Maire yew (Taxus mairei), an evergreen conifer, has high ornamental and medicinal value. The arils of this species has three different colors. However, the variation mechanisms of arils color formation remains unclear. Here, the gene expression and metabolite concentration were profiled for red (RTM), yellow (YTM), and purple (PTM) arils in different developmental stages. A total of 266 flavonoids and 35 carotenoids were identified. The predominant pigments identified in YTM were epiafzelechin, lutein, and β-Cryptoxanthin, while malvidin-3,5-di-O-glucoside and apigenin played crucial roles in PTM. And significant differential expression was observed among the HCT, DFR, LAR, ANS, crtB, NCED, and CCoAOMT genes across different color arils. During the maturation of yellow arils, the upregulation of HCT was strongly correlated with the accumulation of epiafzelechin. The diminished expression of DFR, LAR, and ANS seemed to inhibit the production of delphinidin-3-O-rutinoside. The decrease in crtB expression and concurrent increase in NCED expression potentially regulate the heightened accumulation of lutein. Meanwhile, the accumulation of β-cryptoxanthin appeared seemed to be positively influenced by NCED. As aril turning purple, the decreased expression of CCoAOMT seemed to facilitate the synthesis of apigenin. The substantial upregulation of DFR promoted the production of malvidin-3,5-di-O-glucoside. Additionally, the overexpression of MYBs may plays the important role in regulating the formation of different colored arils. In total, 14 genes were selected for qRT-PCR validation, the results indicated the reliability of the transcriptome sequences data. Our findings could provide valuable insight into the molecular breeding, development, and application of Maire yew resources.

Molecular analysis of structural genes involved in flavonoids biosynthesis in naturally colored cotton

AbstractIn order to facilitate the development of cotton (Gossypium hirsutum L.) strains with colored fiber, the genetic and developmental control of flavonoids in fibers must be understood. The major goal of this study was to determine whether anthocyanidins accumulate in three different natural colored cotton fibers during their development, and to document the expression levels of flavonoid genes necessary for the production of anthocyanidins to determine if differences in expression levels reflect differences in anthocyanidin production. Most flavonoid structural genes were more highly expressed during fiber elongation than before the beginning of secondary cell wall formation. High performance liquid chromatography (HPLC) analyses showed that the abundance of anthocyanidins was higher in brown fibers than in green or white fibers. Anthocyanidins were present at high levels in fibers, starting at 20 d post‐anthesis of fiber development, which is almost at the end of the fiber elongation stage. Brown cotton contains higher delphinidin levels than of green and white cotton. This pattern is consistent with greater expression of anthocyanin pathway genes in brown fibers. In naturally colored cotton, the enzyme Lar (leucoanthocyanidin reductase) converted leucoanthocyanidins to catechin, which are proanthocyanins (PAs) precursor subunits. It is suggested that failure to produce anthocyanins is likely due to a combination of insufficient expression of Ufgt (UDP‐glucose: flavonoid 3‐O‐glucosyltransferase) and high expression of Lar and Anr (anthocyanidin reductase).

Selective expression of CSPG receptors PTPσ and LAR in poorly regenerating reticulospinal neurons of lamprey

Disability following spinal cord injury is due to failure of axon regeneration, which has been ascribed to environmental factors in the central nervous system and a developmental loss of intrinsic growth capacity in neurons. Recently, the receptor-like protein tyrosine phosphatases, protein tyrosine phosphatase σ (PTPσ) and leukocyte common antigen-related phosphatase (LAR), have been identified as specific receptors for the regeneration-inhibiting matrix molecules chondroitin sulfate proteoglycans (CSPGs). After spinal cord transection in lampreys, axons of the large, identified reticulospinal neurons have heterogeneous regenerative abilities. The bad-regenerating neurons also undergo a delayed form of axotomy-induced apoptosis. In the present study, a lamprey genomic database was used to identify homologs of CSPGs, clone PTPσ and LAR, and examine their mRNA expression. CSPG immunoreactivity was increased significantly near the lesion at 2 weeks post transection, and decreased thereafter. Both receptors were expressed selectively in the bad-regenerating neurons and had overlapping cellular distributions. PTPσ was upregulated with age (LAR was not evaluated). By 2 weeks post transection, neurons expressing PTPσ also showed caspase activation, suggesting apoptosis. The probability of axon regeneration for individual identified neurons was negatively correlated with the expression level of PTPσ in both control and spinal cord-transected lampreys. In an animal 7 weeks post transection, regenerated axons were labeled retrogradely from beyond the transection. PTPσ expression and caspase labeling was seen only in neurons whose axon had not regenerated. These results are consistent with a possible role for PTPσ (and LAR) in both retrograde neuronal death and the poor intrinsic regenerative ability of bad-regenerating neurons.

Physiological factors affecting transcription of genes involved in the flavonoid biosynthetic pathway in different rice varieties

Flavonoids play an important role in the grain color and flavor of rice. Since their characterization in maize, the flavonoid biosynthetic genes have been extensively studied in grape, Arabidopsis, and Petunia. However, we are still a long way from understanding the molecular features and mechanisms underlying the flavonoid biosynthetic pathway. The present study was undertaken to understand the physiological factors affecting the transcription and regulation of these genes. We report that the expression of CHI, CHS, DFR, LAR, and ANS, the 5 flavonoid biosynthetic genes in different rice varieties, differ dramatically with respect to the stage of development, white light, and sugar concentrations. We further demonstrate that white light could induce the transcription of the entire flavonoid biosynthetic gene pathway; however, differences were observed in the degrees of sensitivity and the required illumination time. Our study provides valuable insights into understanding the regulation of the flavonoid biosynthetic pathway.

Retrograde BMP Signaling at the Synapse: A Permissive Signal for Synapse Maturation and Activity-Dependent Plasticity

At the Drosophila neuromuscular junction (NMJ), the loss of retrograde, trans-synaptic BMP signaling causes motoneuron terminals to have fewer synaptic boutons, whereas increased neuronal activity results in a larger synapse with more boutons. Here, we show that an early and transient BMP signal is necessary and sufficient for NMJ growth as well as for activity-dependent synaptic plasticity. This early critical period was revealed by the temporally controlled suppression of Mad, the SMAD1 transcriptional regulator. Similar results were found by genetic rescue tests involving the BMP4/5/6 ligand Glass bottom boat (Gbb) in muscle, and alternatively the type II BMP receptor Wishful Thinking (Wit) in the motoneuron. These observations support a model where the muscle signals back to the innervating motoneuron's nucleus to activate presynaptic programs necessary for synaptic growth and activity-dependent plasticity. Molecular genetic gain- and loss-of-function studies show that genes involved in NMJ growth and plasticity, including the adenylyl cyclase Rutabaga, the Ig-CAM Fasciclin II, the transcription factor AP-1 (Fos/Jun), and the adhesion protein Neurexin, all depend critically on the canonical BMP pathway for their effects. By contrast, elevated expression of Lar, a receptor protein tyrosine phosphatase found to be necessary for activity-dependent plasticity, rescued the phenotypes associated with the loss of Mad signaling. We also find that synaptic structure and function develop using genetically separable, BMP-dependent mechanisms. Although synaptic growth depended on Lar and the early, transient BMP signal, the maturation of neurotransmitter release was independent of Lar and required later, ongoing BMP signaling.

Characterization of Flavan-3-ols and Expression of MYB and Late Pathway Genes Involved in Proanthocyanidin Biosynthesis in Foliage of Vitis bellula.

Proanthocyanidins (PAs) are fundamental nutritional metabolites in different types of grape products consumed by human beings. Although the biosynthesis of PAs in berry of Vitis vinifera has gained intensive investigations, the understanding of PAs in other Vitis species is limited. In this study, we report PA formation and characterization of gene expression involved in PA biosynthesis in leaves of V. bellula, a wild edible grape species native to south and south-west China. Leaves are collected at five developmental stages defined by sizes ranging from 0.5 to 5 cm in length. Analyses of thin layer chromatography (TLC) and high performance liquid chromatography-photodiode array detector (HPLC-PAD) show the formation of (+)-catechin, (−)-epicatechin, (+)-gallocatechin and (−)-epigallocatechin during the entire development of leaves. Analyses of butanol-HCl boiling cleavage coupled with spectrometry measurement at 550 nm show a temporal trend of extractable PA levels, which is characterized by an increase from 0.5 cm to 1.5 cm long leaves followed by a decrease in late stages. TLC and HPLC-PAD analyses identify cyanidin, delphinidin and pelargonidin produced from the cleavage of PAs in the butanol-HCl boiling, showing that the foliage PAs of V. bellula include three different types of extension units. Four cDNAs, which encode VbANR, VbDFR, VbLAR1 and VbLAR2, respectively, are cloned from young leaves. The expression patterns of VbANR and VbLAR2 but not VbLAR1 and VbDFR follow a similar trend as the accumulation patterns of PAs. Two cDNAs encoding VbMYBPA1 and VbMYB5a, the homologs of which have been demonstrated to regulate the expression of both ANR and LAR in V. vinifera, are also cloned and their expression profiles are similar to those of VbANR and VbLAR2. In contrast, the expression profiles of MYBA1 and 2 homologs involved in anthocyanin biosynthesis are different from those of VbANR and VbLAR2. Our data show that both ANR and LAR branches are involved in PA biosynthesis in leaves of V. bellula.

Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport

The colour of the red wine is essentially due to the release of anthocyanins from the red skin of grape berries during the process of wine making. Anthocyanins are synthesized during ripening of the berries under the control of VvMYBA1 transcription factor that controls the expression of UFGT. In order to identify the whole set of downstream regulated genes, we targeted constitutive ectopic expression of VlmybA1-2 into grapevine hairy roots and plants. The ectopic expression of VlmybA1-2 triggered de novo production and storage of anthocyanins in all transgenic vegetative organs, leading to a very intense red coloration, and did not interfere with proanthocyanidin (PA) biosynthesis. The ectopic red pigmentation was due to the accumulation of anthocyanins in vacuoles and anthocyanin vacuolar inclusion (AVIs) in all organs but only in specific tissues. A transcriptomic analysis using a 14 K oligoarray revealed that the ectopic expression of VlmybA1-2 activated only few genes, most of which are involved in both PA and anthocyanin biosynthesis, while the expression of BAN and LAR (two specific genes of the PA biosynthesis pathway) was unaffected. Among these, 4 genes emerged given the amplitude of their up-regulation, quantitatively similar to VlmybA1-2 itself. In addition to the previously described UFGT, this set comprised an isogen of GST, an O-methyltransferase, both of which are supposed to play a role in the anthocyanin biosynthesis pathway, as well as a candidate gene putatively involved in the vacuolar anthocyanin transport in grapevine (anthoMATE). Together, these results suggest that MybA1 activates the last steps of anthocyanin synthesis and transport through the regulation of a narrow, specific spectrum of genes regulated as a cluster.

Nerfin-1 is required for early axon guidance decisions in the developing Drosophila CNS

Many studies have focused on the mechanisms of axon guidance; however, little is known about the transcriptional control of the navigational components that carryout these decisions. This report describes the functional analysis of Nerfin-1, a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. We show that the neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development nor in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Our studies also show that Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch, genes whose encoded proteins participate in these early navigational events.

Transgenic Overexpression of Protein-tyrosine Phosphatase 1B in Muscle Causes Insulin Resistance, but Overexpression with Leukocyte Antigen-related Phosphatase Does Not Additively Impair Insulin Action

Previous studies implicate protein-tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related phosphatase (LAR) as negative regulators of insulin signaling. The expression and/or activity of PTP1B and LAR are increased in muscle of insulin-resistant rodents and humans. Overexpression of LAR selectively in muscle of transgenic mice causes whole body insulin resistance. To determine whether overexpression of PTP1B also causes insulin resistance, we generated transgenic mice overexpressing human PTP1B selectively in muscle at levels similar to those observed in insulin-resistant humans. Insulin-stimulated insulin receptor (IR) tyrosyl phosphorylation and phosphatidylinositol 3'-kinase activity were impaired by 35% and 40-60% in muscle of PTP1B-overexpressing mice compared with controls. Insulin stimulation of protein kinase C (PKC)lambda/zeta activity, which is required for glucose transport, was impaired in muscle of PTP1B-overexpressing mice compared with controls, showing that PTP1B overexpression impairs activation of these PKC isoforms. Furthermore, hyperinsulinemic-euglycemic clamp studies revealed that whole body glucose disposal and muscle glucose uptake were decreased by 40-50% in PTP1B-overexpressing mice. Overexpression of PTP1B or LAR alone in muscle caused similar impairments in insulin action; however, compound overexpression achieved by crossing PTP1B- and LAR-overexpressing mice was not additive. Antibodies against specific IR phosphotyrosines indicated overlapping sites of action of PTP1B and LAR. Thus, overexpression of PTP1B in vivo impairs insulin sensitivity, suggesting that overexpression of PTP1B in muscle of obese humans and rodents may contribute to their insulin resistance. Lack of additive impairment of insulin signaling by PTP1B and LAR suggests that these PTPs have overlapping actions in causing insulin resistance in vivo.

Cell-Autonomous and -Nonautonomous Functions of LAR in R7 Photoreceptor Axon Targeting

During Drosophila visual system development, photoreceptors R7 and R8 project axons to targets in distinct layers of the optic lobe. We show here that the LAR receptor tyrosine phosphatase is required in the eye for correct targeting of R7 axons. In LAR mutants, R7 axons initially project to their correct target layer, but then retract to the R8 target layer. This targeting defect can be fully rescued by transgenic expression of LAR in R7, and partially rescued by expression of LAR in R8. The phosphatase domains of LAR are required for its activity in R7, but not in R8. These data suggest that LAR can act both as a receptor in R7, and as a ligand provided by R8. Genetic interactions implicate both Enabled and Trio in LAR signal transduction.

Receptor protein tyrosine phosphatases regulate retinal ganglion cell axon outgrowth in the developing Xenopus visual system.

Receptor protein tyrosine phosphatases (RPTPs) are regulators of axon outgrowth and guidance in a variety of different vertebrate and invertebrate systems. Three RPTPs, CRYP-alpha, PTP-delta, and LAR, are expressed in overlapping but distinct patterns in the developing Xenopus retina, including expression in retinal ganglion cells (RGCs) as they send axons to the tectum (Johnson KG, Holt CE. 2000. Expression of CRYP-alpha, LAR, PTP-delta, and PTP-rho in the developing Xenopus visual system. Mech Dev 92:291-294). In order to examine the role of these RPTPs in visual system development, putative dominant negative RPTP mutants (CS-CRYP-alpha, CS-PTP-delta, and CS-LAR) were expressed either singly or in combination in retinal cells. No effect was found on either retinal cell fate determination or on gross RGC axon guidance to the tectum. However, expression of these CS-RPTP constructs differentially affected the rate of RGC axon outgrowth. In vivo, expression of all three CS-RPTPs or CS-PTP-delta alone inhibited RGC axon outgrowth, while CS-LAR and CS-CRYP-alpha had no significant effect. In vitro, expression of CS-CRYP-alpha enhanced neurite outgrowth, while CS-PTP-delta inhibited neurite outgrowth in a substrate-dependent manner. This study provides the first in vivo evidence that RPTPs regulate retinal axon outgrowth.

Within the hemopoietic system, LAR phosphatase is a T cell lineage-specific adhesion receptor-like protein whose phosphatase activity appears dispensable for T cell development, repertoire selection and function

Expression of the receptor-type tyrosine phosphatase LAR was studied in cells of the murine hemopoietic system. The gene is expressed in all cells of the T cell lineage but not in cells of any other hemopoietic lineage and the level of expression in T cells is developmentally regulated. The CD4(-)8(-)44(+) early thymic immigrants and mature (CD4(+)8(-)/CD4(-)8(+)) thymocytes and T cells express low levels, whereas immature (CD4(-)8(-)44(-) and CD4(+)8(+)) thymocytes express high levels of LAR. Among bone marrow cells only uncommitted c-kit(+)B220(+)CD19(-) precursors, but not B cell lineage committed c-kit(+)B220(+)CD19(+) precursors, express low levels of LAR. In contrast to the c-kit(+)B220(+)CD19(+) pre-BI cells from normal mice, counterparts of pre-BI cells from PAX-5-deficient mice express LAR, indicating that PAX-5-mediated commitment to the B cell lineage results in suppression of LAR. During differentiation of PAX-5-deficient pre-BI cell line into non-T cell lineages, expression of LAR is switched off, but it is up-regulated during differentiation into thymocytes. Thus, within the hemopoietic system, LAR appears to be a T cell lineage-specific receptor-type phosphatase. However, surprisingly, truncation of its phosphatase domains has no obvious effect on T cell development, repertoire selection or function.

Expression of Protein Tyrosine Phosphatases in the Preimplantation Mouse Embryos

The expression of protein tyrosine phosphatases (PTPs) was examined in preimplantation mouse embryos. PTPs expressed in the various developmental stages, including unfertilized oocytes, 2-cell, 4-cell, morula and blastocyst stages, were identified by reverse transcription of mRNA followed by a polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers corresponding to the conserved catalytic domain of PTP. Sequencing of cDNA clones obtained by RT-PCR revealed the expression of mRPTPB, SHP-1, PTPT9, SHP-2 and LAR. The changes in the expression levels of these PTPs during preimplantation development were examined by RT-PCR using each gene specific primers. The results showed that their expression levels dynamically changed in different manners among PTPs, suggesting that PTPs play roles in the regulation of growth and differentiation during preimplantation development.

Specific inhibition of FGF-induced MAPK activation by the receptor-like protein tyrosine phosphatase LAR.

LAR is a widely expressed receptor-like protein tyrosine phosphatase that is implicated in regulation of intracellular signaling triggered by both cell adhesion and peptide growth factors. Genetic studies revealed that LAR regulates neuron axon path finding in Drosophila and mammary gland epithelial cell differentiation in mice. The molecular mechanism underlying the tissue specific function of LAR has not been clearly understood. We investigated the role and mechanism of LAR in peptide growth factors EGF and FGF signaling in human tissue culture cells in which the expression of LAR is under the control of an inducible promoter. We found that although both EGF and FGF induce activation of mitogen-activated protein kinase (MAPK), LAR only inhibits FGF-induced MAPK activation. LAR does not interact directly with the peptide growth factor receptors, since the ligand-induced autophosphorylation of growth factor receptors was not affected by induction of LAR. The specific effect of LAR on FGF-induced MAPK activation appeared to be mediated by specific inhibition of the phosphorylation of two signal transducers that act downstream of the FGF receptor, FRS2 and a 180 kDa protein, and by prevention of their interaction with the adaptor protein GRB2. In contrast, LAR selectively inhibited the epidermal growth factor (EGF)-induced phosphorylation of p130CAS and the formation of the complex between p130CAS and GRB2 but this effect did not influence the activation of MAPK by EGF. These data suggest that LAR and similar receptor-like protein tyrosine phosphatases may contribute to the regulation of transmembrane signaling by selectively inhibiting the tyrosine phosphorylation of specific signal transducers that act downstream of the plasma membrane-associated tyrosine kinases. The consequent inhibition of the formation of signaling complexes by these proteins may contribute to the specificity of the signals generated by specific peptide growth factors as well as extracellular matrix proteins.

Changes of Expressions of Phosphotyrosine Phosphatases in Rat Hepatocellular Carcinoma Induced by 3′-Methyl-4-dimethylamino-azobenzene

Using rats with hepatocellular carcinoma induced by 3′-methyl-4-dimethylamino-azobenzene (3′-MeDAB), we evaluated the expression of a protein tyrosine phosphatase (PTPase) in the tumor region, non-tumorous region and control rat liver. The expression of SHPTP2 increased 4.1 fold (p<0.05) in mRNA, 2.1 fold (p<0.01) in cytosol fraction, and 5.1 fold (p<0.05) in membrane fraction, respectively, at a protein level in the tumor region compared with control liver. The expression of other phosphatases, LAR, LRP, and PTPase1B, did not change significantly. SHPTP2 phosphatase activity in the tumor region from rats also increased compared with the control, suggesting that an increase of this activity may parallel the expression of SHPTP2. This increase of expression of SHPTP2 may contribute to the progression of hepatocellular carcinoma in this rat model.

Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region.

The human transmembrane molecule LAR is a protein tyrosine phosphatase (PTPase) with a cell adhesion molecule-like extracellular receptor region. The structure of LAR hinted at its involvement in the regulation of tyrosine phosphorylation through cell-cell or cell-matrix interactions. We show here that LAR is expressed on the cell surface as a complex of two non-covalently associated subunits derived from a proprotein. The LAR E-subunit contains the cell adhesion molecule-like receptor region, while the LAR P-subunit contains a short segment of the extracellular region, the transmembrane peptide and the cytoplasmic PTPase domains. Proprotein processing occurs intracellularly. Analysis of LAR mutants suggested that cleavage occurs in the LAR extracellular region at a paired basic amino acid site by a subtilisin-like endoprotease. A single amino acid substitution at this site blocked LAR proprotein cleavage. The LAR E-subunit is shed during cell growth, suggesting that LAR receptor shedding may be a mechanism for regulating PTPase function. The use of immunohistochemistry techniques on human tissues demonstrated the expression of LAR by various cell lineages, including epithelial cells, smooth muscle cells and cardiac myocytes. The LAR gene is mapped to chromosome 1, region p32-33, which contains candidate tumor suppressor genes.