Tissue-specific expression of two alternatively spliced insulin receptor mRNAs in man

Two previously reported insulin receptor cDNA sequences differ by 36 base pairs (bp) in the distal alpha-subunit, suggesting that alternative mRNA splicing within the coding region may occur (two insulin receptor isoforms). We developed a quantitative modification of the polymerase chain reaction technique in order to detect and characterize differential mRNA splicing at this site within the distal alpha-subunit. Using RNA derived from a variety of human cell types, we detected two polymerase chain reaction-amplified cDNA species reflecting the presence or absence of the above 36 nucleotides. Identity of the two cDNA species was confirmed by Southern blots, the use of a BANI restriction site present only in the 36 base pair segment and dideoxy sequencing. The relative expression of the two mRNA forms varied markedly in a tissue-specific manner. Buffy coat leukocytes and Epstein-Barr virus-transformed lymphocytes express only the shorter mRNA. Placenta expresses both species equally; muscle, isolated adipocytes and cultured fibroblasts express somewhat more of the longer mRNA (relative ratios of mRNA abundance of 1.51, 3.18, and 2.77, respectively); liver expresses mostly the longer mRNA (relative ratio of 9.8). In RNA derived from cultured and fresh cells from patients with several states of insulin resistance, the relative expression of the two mRNA species was similar to results obtained with comparable normal tissues. Although the functional significance of alternative splicing of the insulin receptor mRNA is unknown, differential expression of these two receptor mRNAs may provide a structural basis for previously observed tissue-specific differences in insulin binding and action.     

Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced

Several ribosomal protein families contain paralogues whose roles may be equivalent or specialized to include extra-ribosomal functions. RpL22e family members rpL22 and rpL22-like are differentially expressed in Drosophila melanogaster: rpL22-like mRNA is gonad specific whereas rpL22 is expressed ubiquitously, suggesting distinctive paralogue functions. To determine if RpL22-like has a divergent role in gonads, rpL22-like expression was analysed by qRT-PCR and western blots, respectively, showing enrichment of rpL22-like mRNA and a 34 kDa (predicted) protein in testis, but not in ovary. Immunohistochemistry of the reproductive tract corroborated testis-specific expression. RpL22-like detection in 80S/polysome fractions from males establishes a role for this tissue-specific paralogue as a ribosomal component. Unpredictably, expression profiles revealed a low abundant, alternative mRNA variant (designated 'rpL22-like short') that would encode a novel protein lacking the C-terminal ribosomal protein signature but retaining part of the N-terminal domain. This variant results from splicing of a retained intron (defined by non-canonical splice sites) within rpL22-like mRNA. Polysome association and detection of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing of rpL22-like generates structurally distinct protein products: ribosomal component RpL22-like and a novel protein with a role distinct from RpL22-like.     

The c-Jun N-terminal kinase 1 activity is differentially regulated by specific mechanisms during apoptosis

We show here that JNK1 activity is rapidly up-regulated and prolonged by specific mechanisms during apoptosis induced by paclitaxel- or ginsenoside-Rh2 in SK-HEP-1 cells. The early phase of JNK1 activation is prevented in cells expressing the dominant negative SEK1 mutant, although this JNK1 perturbation does not prevent apoptotic cell death. The later phase of JNK1 activation, which is temporally coincided with caspase-dependent cleavage of JNK1-associated p21(WAF1/CIP1), is efficiently prevented by expressing p21D112N, an uncleavable mutant of p21(WAF1/CIP1) and this perturbation of JNK1 activation results in prevention of apoptosis. The later JNK1 activation and apoptotic progression are also prevented by co-treatments of cells with rottlerin, a PKC-delta inhibitor or z-VAD-fmk, a pan caspase inhibitor. We also provide evidence that apoptotic cell death is significantly promoted in cells expressing JNK1, while this apoptotic cell death is effectively suppressed in cells expressing the dominant negative JNK1 mutant (DN-JNK1) or JBD, a JNK inhibitor protein. Thus, the later phase of JNK1 activation, which is linked to a caspase-dependent mechanism that requires PKC-delta activity, is associated with the induction of apoptosis, while the early JNK1 activation that is associated with a SEK1-mediated mechanism is not directly involved in apoptotic progression.     

Biological activity of Manduca sexta allatotropin-like peptides,predicted products of tissue-specific and developmentally regulated alternatively spliced mRNAs

The insect neuropeptide, allatotropin (Manse-AT), exerts multiple functions including the stimulation of juvenile hormone (JH) biosynthesis in adults and the inhibition of active ion transport across the midgut epithelium of feeding larvae. The Manse-AT gene is expressed in multiple regions of the nervous system as three mRNAs that differ by alternative splicing. The specific mRNA isoform present differs in a tissue- and developmental-specific manner thus providing a mechanism for the regulated production of peptides specific to each isoform. These peptides are predicted to include three allatotropin-like (Manse-ATL) peptides that exhibit limited structural identity to Manse-AT and overlapping biological activities.     

Brain-specific interleukin-1 receptor accessory protein in sleep regulation

Interleukin (IL)-1β is involved in several brain functions, including sleep regulation. It promotes non-rapid eye movement (NREM) sleep via the IL-1 type I receptor. IL-1β/IL-1 receptor complex signaling requires adaptor proteins, e.g., the IL-1 receptor brain-specific accessory protein (AcPb). We have cloned and characterized rat AcPb, which shares substantial homologies with mouse AcPb and, compared with AcP, is preferentially expressed in the brain. Furthermore, rat somatosensory cortex AcPb mRNA varied across the day with sleep propensity, increased after sleep deprivation, and was induced by somnogenic doses of IL-1β. Duration of NREM sleep was slightly shorter and duration of REM sleep was slightly longer in AcPb knockout than wild-type mice. In response to lipopolysaccharide, which is used to induce IL-1β, sleep responses were exaggerated in AcPb knockout mice, suggesting that, in normal mice, inflammation-mediated sleep responses are attenuated by AcPb. We conclude that AcPb has a role in sleep responses to inflammatory stimuli and, possibly, in physiological sleep regulation.     

Expression of major HDL-associated antioxidant PON-1 is gender dependent and regulated during inflammation

Paraoxonase 1, an HDL-associated enzyme that confers antioxidant activity on HDL, and its activity in serum have been correlated with protection against atherosclerosis, an oxidative disease. However, serum PON-1 activity is highly variable and its regulation is complex, involving both genetic and environmental factors. It is influenced by gender and inflammation, two important factors in atherosclerosis. Serum PON-1 activity has been shown to be lower in male mice and is decreased in male Syrian hamster during inflammation. Here we show that male mice had lower hepatic PON-1 mRNA that increased by 170% after castration. Our data also suggested that this effect was testes but not plasma testosterone dependent. Ovariectomy had no effect on PON-1 mRNA in female mice. LPS caused hepatic PON-1 mRNA to decrease further in male mice, and to increase moderately in female mice. Anti-inflammatory dexamethasone enhanced PON-1 mRNA level by 2-fold in male and female LPS-treated mice, and increased PON-1 expression by 8-fold in Hepa cell, a mouse hepatoma cell line. Therefore, antioxidant PON-1 is regulated at the mRNA level in a gender-specific manner by proinflammatory LPS and anti-inflammatory dexamethasone.     

Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1).

The gene (HSD-1) coding a human sperm membrane protein (hSMP-1) was isolated from a human testis cDNA expression library using antibodies found in the serum of an infertile woman. HSD-1 was localized to a single locus on chromosome 9 and assigned to band 9p12-p13 by fluorescent in situ hybridization (FISH) mapping and DAPI (4,6-diamidino-2-phenylindole) banding, using rat/human somatic cell hybrids and metaphase chromosomes of human lymphocytes. In rescreening a testis lambdagt10 cDNA expression library, the full-length cDNA (HSD-1) and several truncated cDNAs with heterologous regions were isolated from positive clones. The heterology consisted of deletion, insertion and alteration of the 5'-end. These heterologous truncated fragments may be produced by alternative splicing of mRNAs. Two recombinant prokaryotic expression vectors were constructed with one of the heterologous fragment (clone #26) with and without the alternative 5'-end. Escherichia coli transfected with the construct containing the alternative 5'-end failed to produce the recombinant product, whereas those transfected with the vector lacking the 5'-end produced hSMP-1. DNASIS analysis of the structure of #26 mRNA suggests that the 5'-end has a stable secondary configuration that may maintain the mRNA in an inactivated state, whereby hindering its translation and preventing the expression of the gene.     

Contribution of interleukin-1 receptor accessory protein B to interleukin-1 actions in neuronal cells

Interleukin (IL)-1 is an important neuroimmunomodulator and a key mediator of inflammation during brain disorders. It acts on neuronal and glial cells via binding to the IL-1 type 1 receptor and IL-1 receptor accessory protein (IL-1RAcP). More recently, a neuronal-specific isoform of IL-1RAcP, named IL-1RAcPb, has been identified. Our aim was to determine the role of IL-1RAcPb in IL-1 actions in neuronal and glial cells, and to further explore the signaling mechanisms of IL-1 in neurons. We found that IL-1RAcPb deletion had no effect on IL-1α- and IL-1β-induced activation of the extracellular signal-regulated kinase 1/2 or IL-6 release in glial cultures, although IL-6 release in response to high IL-1α concentration (30 IU/ml) was significantly reduced. We identified the p38 kinase as a key signaling element in IL-1α- and IL-1β-induced IL-6 synthesis and release in neuronal cultures. IL-1RAcPb deletion had no effect on IL-1α- and IL-1β-induced IL-6 release in neurons, but significantly reduced IL-1α- but not IL-1β-induced p38 phosphorylation. Our data demonstrate that the p38 signaling pathway plays an important role in IL-1 actions in neurons, and that IL-1RAcP may regulate some, but not all, neuronal activities in response to IL-1α.     

Insulin-like growth factor binding protein 5 and type-1 insulin-like growth factor receptor are differentially regulated during apoptosis in cerebellar granule cells

Neuronal apoptosis is considered to play a significant role in several neuropathological conditions. However, the molecular mechanisms underlying neuronal apoptosis are poorly understood. Insulin-like growth factor (IGF) signalling is considered to be an important regulator of neuronal differentiation, survival and apoptosis. We have examined the expression of two members of the IGF system, insulin-like growth factor binding protein 5 (IGFBP-5) and the type-1 IGF receptor (IGF1R), during apoptosis of rat cerebellar granule cells (CGCs) in vitro. We describe a prominent downregulation of IGFBP-5 mRNA and protein expression. We also show that IGF-I increases IGFBP-5 expression in CGCs and that the downregulation of IGFBP-5 mRNA can be suppressed by inhibiting mRNA synthesis with actinomycin D. The expression of IGF1R mRNA showed a transient upregulation during potassium chloride (KCl) deprivation induced apoptosis, in contrast to the IGF1R protein level, which was downregulated during KCl deprivation. Our results provide insight into the expression of IGF-related genes during neuronal apoptosis, and indicate that they mediate a protective response to the withdrawal of trophic stimulation. It seems that the expression of IGFBP-5 and IGF1R is regulated to maximize the availability of IGF and the activity of IGF-triggered survival signalling.