Alternative splicing of GTBP in normal human tissues

The relationship between serum hepatitis Be antigen (HBeAg) and serum hepatitis B virus (HBV) DNA determined by two commercially available assays was examined in 345 Chinese patients with chronic HBV infection. HBV DNA was detected by these commercial assays in 85% of the HBeAg-positive patients. Discrepancies between test results were found to occur when serum HBV DNA levels were low (< 5 pg ml-1 for the Abbott Genostics and < 100 MEq ml-1 for Chiron Quantiplex assays). An equation for the conversion between results generated by these two assays was derived, which was found to be very similar to the equation recently described by Kapke et al.     

Alternative splicing of human NrCAM in neural and nonneural tissues

HEC1A endometrial cancer cells express the wild-type form of the estrogen receptor (ER) and 17beta-estradiol (E2) induces proliferation of these cells. In contrast, tamoxifen only causes a minimal increase (<20%) in cell proliferation. In HEC1A cells transiently transfected with the C3-Luc plasmid derived from the complement C3 gene, both E2 and tamoxifen exhibited ER agonist activity and tamoxifen was also a partial antagonist for this response. The relative ER agonist/antagonist activities of E2, tamoxifen and ICI 182,780 were also investigated in HEC1A1 cells transiently transfected with two E2-responsive plasmids, pCATHD-CAT and pCKB-CAT which contain 5'-promoter inserts from the cathepsin D and creatine kinase B genes, respectively. The results showed that E2 and tamoxifen induced reporter gene activity in cells transiently transfected with both constructs. ICI 182,780 exhibited partial ER agonist activity only in cells transiently transfected with pCKB-CAT and antagonized E2-induced reporter gene activity using both the CKB- and CATHD-derived constructs. These results demonstrate that HEC1A endometrial cancer cells are E2-responsive and represent a useful cell culture model for understanding hormone/antihormone-induced endometrial cell responses.     

Alternative splicing of CYP2D mRNA in human breast tissue

Cerebrovascular arteriovenous malformations (AVMs) display abnormal vascular development and dysautoregulation of blood flow. Genetic mechanisms that contribute to the pathogenesis and phenotype of cerebral AVMs are unknown. As a first step in understanding the pathophysiology of AVMs, the authors investigated the hypothesis that endothelial dysfunction-specifically, deregulation of endothelin-1 (ET-1) secretion-contributes to the abnormal vascular phenotype and the lack of hemodynamic autoregulation elaborated by these lesions. Endothelin-1 peptide and preproendothelin-1 (ppET1) messenger RNA were not detected in the intranidal vasculature of all 17 patients with AVMs studied, but were prominently expressed in human control subjects with normal cerebrovasculature (p < 0.01). Although AVM vasculature lacked ET-1, its expression was prominent in vasculature distant from these lesions, suggesting local repression of the ppET-1 gene. Local repression of ET-1 was specific to AVMs; ET-1 in vascular malformations of patients with Sturge-Weber disease was actually elevated compared to normal controls (p < 0.01). Repression of the ppET-1 gene was an intrinsic phenotype of AVM endothelial cells and was not due to factors in the AVM microenvironment. The authors also showed that ETA receptor expression was low in AVM vasculature compared to normal controls. Together, these results demonstrate that the ppET-1 gene is locally repressed in AVM lesions and suggest a role for abnormal ppET-1 gene regulation in the pathogenesis and clinical sequelae of cerebral AVMs.     

Alternative splicing of LTBP-3

LTBPs bind the 100-kDa latent TGF-beta complex and thereby regulate TGF-beta assembly, tissue localization, and function. However, the 100-kDa complex is not always associated with LTBP, and, conversely, evidence suggests that LTBP has a distinct role in the extracellular matrix. As yet, there are no data to explain how the binding interaction between LTBP and the 100-kDa complex is regulated. This report provides the first direct evidence of alternative splicing of an LTBP gene. Two alternative splice sites in the mouse LTBP-3 gene have been identified based on in vivo and in vitro studies. Alternative splicing at one site in particular was found to disrupt a structural motif involved in the binding interaction with the 100-kDa latent TGF-beta complex. Therefore, alternative splicing may represent a molecular mechanism by which the uncomplexed form of LTBP-3 is produced, and, as a corollary, by which the 100-kDa latent TGF-beta 1 complex is produced.     

Molecules in focus: diacylglycerol kinase

Recent observations suggest that diacylglycerol kinase (DGK) is one of the key enzymes involved in the regulation of signal transduction. It attenuates protein kinase C activity and cell cycle progression of T-lymphocytes, through controlling the intracellular levels of the second messengers, diacylglycerol and phosphatidic acid. To date, eight DGK isozymes containing characteristic zinc finger structures in common have been identified. Type I DGKs (alpha, beta and gamma) contain EF-hand motifs that contribute to the calcium-dependent activities of this type of DGK. A pleckstrin homology and/or an EPH C-terminal tail homology domains are found in type II isozymes (DGK delta and eta). DGK epsilon represents a third type of DGK that selectively phosphorylates arachidonate-containing diacylglycerol. DGK zeta (type IV) and DGK theta (type V) contain four tandem ankyrin repeats and a Ras-associating domain, respectively.     

Alternative splicing of a 48-nucleotide exon generates two isoforms of the human calcitonin receptor

A portion of the human calcitonin receptor (hCTR) gene corresponding to the region of the porcine gene at which alternative splicing generates two CTR isoforms was isolated by polymerase chain reaction amplification of placental DNA. In contrast to the porcine CTR gene, in which two acceptor sites in exon 8 are separated by 48 nucleotides, we found a distinct 48-nucleotide exon in the hCTR gene that is present approximately 6400 basepairs from the up-stream exon, which corresponds to porcine exon 7, and approximately 1100 basepairs from the down-stream exon, which corresponds to porcine exon 8. Splicing of this exon accounts for the two isoforms of hCTR, containing or not containing a 16-amino acid insertion in the first putative intracellular loop. A region similar to the intron 7-exon junction in the porcine CTR gene is present in the human gene, but contains four extra nucleotides that shift the reading frame. Using probes derived from these introns in somatic cell and in situ hybridization analyses, we assigned the CTR gene to human chromosome band 7q21.2-q21.3. Thus, human and porcine species have evolved distinct mechanisms to generate two similar CTR isoforms.     

Alternative splicing events in the coding region of the cytochrome P450 aromatase gene in male rat germ cells

The metabolic and muscle blood flow response in recovery from exercise is dependent on the type and the duration of the exercise. Immediately after both intense static and dynamical exercise blood flow to the exercised muscles increases suggesting that blood flow is mechanically hindered by muscle contraction. After the initial rise (seconds) muscle blood flow decreases at a moderate rate and the time to reach resting flow levels varies from seconds to more than 30 min. It is unclear as to what causes the elevated blood flow during recovery. A mismatch between the time course of changes in blood flow and oxygen uptake suggests that the blood flow is not directly regulated by the need of oxygen in the exercised muscles. The hyperaemic response may be linked to locally released factors, such as ions and metabolites. However, the signal by which the blood flow is elevated remains unknown. After exercise both pulmonary and muscle oxygen uptake decrease rapidly, but can remain above resting levels for several hours. Resynthesis of substrates such as CP, ATP and glycogen cannot account for the entire excessive post-exercise oxygen uptake (EPOC) in the exercised muscles and the cause of the elevated muscle oxygen uptake in recovery from exercise remains to be assessed.     

Protein kinase C regulates the nuclear localization of diacylglycerol kinase-zeta

Diacylglycerol kinases (DGKs) terminate signalling from diacylglycerol by converting it to phosphatidic acid. Diacylglycerol regulates cell growth and differentiation, and its transient accumulation in the nucleus may be particularly important in this regulation. Here we show that a fraction of DGK-zeta is found in the nucleus, where it regulates the amount of nuclear diacylglycerol. Reducing nuclear diacylglycerol levels by conditional expression of DGK-zeta attenuates cell growth. The nuclear-localization signal of DGK-zeta is located in a region that is homologous to the phosphorylation-site domain of the MARCKS protein. This is, to our knowledge, the first evidence that this domain, which is a major target for protein kinase C, can localize a protein to the nucleus. Two isoforms of protein kinase C, but not others, regulate the localization of DGK-zeta. Our results define a cycle in which diacylglycerol activates protein kinase C, which then regulates the metabolism of diacylglycerol by alternating the intracellular location of DGK-zeta. This may be a general mechanism to control mitogenic signals that depend on nuclear diacylglycerol.     

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) in human prostate cancer

Progression of prostate cancer from an androgen sensitive to androgen insensitive tumor has previously been shown to be accompanied by a change in alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) in a rat model of prostate cancer. This change results in loss of the FGF-R2(IIIb) isoform and predominant expression of the FGF-R2(IIIc) isoform. We sought to determine whether this change in FGF-R2 splicing is also associated with androgen insensitivity in human prostate tumors. We analysed three well characterized human prostate cancer cell lines and three metastatic prostate tumors which have been maintained as xenografts in nude mice. One of the cell lines, LNCaP, and two of the xenografts, DUKAP-1 and DUKAP-2, have been characterized as androgen sensitive, whereas two of the cell lines, DU-145 and PC-3, and one of the xenografts, DU9479, display androgen independent growth. Using an RT-PCR based assay, we demonstrated that progressive loss of the FGF-R2(111b) isoform correlated with androgen insensitivity in these human prostate cancer models. These findings lend support to the hypothesis that that loss of FGF-R2(IIIb) may be one step in a series of events which lead to progression of human prostate cancer.     

Exercise stimulates the mitogen-activated protein kinase pathway in human skeletal muscle

Physical exercise can cause marked alterations in the structure and function of human skeletal muscle. However, little is known about the specific signaling molecules and pathways that enable exercise to modulate cellular processes in skeletal muscle. The mitogen-activated protein kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. We tested the hypothesis that a single bout of exercise activates the MAPK signaling pathway. Needle biopsies of vastus lateralis muscle were taken from nine subjects at rest and after 60 min of cycle ergometer exercise. In all subjects, exercise increased MAPK phosphorylation, and the activity of its downstream substrate, the p90 ribosomal S6 kinase 2. Furthermore, exercise increased the activities of the upstream regulators of MAPK, MAP kinase kinase, and Raf-1. When two additional subjects were studied using a one-legged exercise protocol, MAPK phosphorylation and p90 ribosomal S6 kinase 2, MAP kinase kinase 1, and Raf-1 activities were increased only in the exercising leg. These studies demonstrate that exercise activates the MAPK cascade in human skeletal muscle and that this stimulation is primarily a local, tissue-specific phenomenon, rather than a systemic response to exercise. These findings suggest that the MAPK pathway may modulate cellular processes that occur in skeletal muscle in response to exercise.     

Smooth muscle alternative splicing induced in fibroblasts by heterologous expression of a regulatory gene

Alternative splicing is a common mechanism for regulating gene expression in different cell types. In order to understand this important process, the trans-acting factors that enforce the choice of particular splicing pathways in different environments must be identified. We have used the rat alpha-tropomyosin gene as a model system of tissue-specific alternative splicing. Exon 3 of alpha-tropomyosin is specifically inhibited in smooth muscle cells allowing the alternative inclusion of exon 2. We have used a novel gene transfer and selection strategy to detect a gene whose expression in fibroblasts is sufficient to switch them to smooth muscle-specific splicing of alpha-tropomyosin and also alpha-actinin. Extracts from the regulating fibroblasts contain an apparently novel 55 kDa protein which binds to RNA elements required for regulation of tropomyosin splicing. This protein is not detected in extracts of non-regulating cells and is therefore a strong candidate cell-specific splicing regulator. These experiments advance our understanding of smooth muscle splicing regulation as well as establishing a means for direct cloning of tissue-specific splicing regulators which have so far been refractory to biochemical analysis.     

Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats

The fibronectin EIIIB exon is alternatively spliced in a cell-type-specific manner, and TGCATG repeats in the intron downstream of EIIIB have been implicated in this regulation. Analysis of the intron sequence from several vertebrates shows that the pattern of repeats in the 3' half of the intron is evolutionarily conserved. Point mutations in certain highly conserved repeats greatly reduce EIIIB inclusion, suggesting that a multicomponent complex may recognize the repeats. Expression of the SR protein SRp40, SRp20, or ASF/SF2 stimulates EIIIB inclusion. Studies of the interplay between mutations in the repeats and SRp40-stimulated inclusion suggest that the repeats are recognized in many, if not all, cell types, and that EIIIB inclusion may be regulated by quantitative changes in multiple factors.