Differential Subcellular Distributions and Trafficking Functions of hnRNP A2/B1 Spliceoforms

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.     

Exon selection in alpha-tropomyosin mRNA is regulated by the antagonistic action of RBM4 and PTB

RNA-binding motif protein 4 (RBM4) has been implicated in the regulation of precursor mRNA splicing. Using differential display analysis, we identified mRNAs that associate with RBM4-containing messenger RNPs in vivo. Among these mRNAs, alpha-tropomyosin (alpha-TM) is known to exhibit a muscle cell type-specific splicing pattern. The level of the skeletal muscle-specific alpha-TM mRNA isoform partially correlated with that of RBM4 in human tissues examined and could be modulated by ectopic overexpression or suppression of RBM4. These results indicated that RBM4 directly influences the expression of the skeletal muscle-specific alpha-TM isoform. Using minigenes, we demonstrated that RBM4 can activate the selection of skeletal muscle-specific exons, possibly via binding to intronic pyrimidine-rich elements. By contrast, the splicing regulator polypyrimidine tract binding protein (PTB) excluded these exons; moreover, RBM4 antagonized this PTB-mediated exon exclusion likely by competing with PTB for binding to a CU-rich element. This study suggests a possible mechanism underlying the regulated alternative splicing of alpha-TM by the antagonistic splicing regulators RBM4 and PTB.     

Tissue-specific distributions of alternatively spliced human PECAM-1 isoforms

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule that is highly expressed on the surface of endothelial cells and some hematopoietic cells. Its cytoplasmic domain is encoded by multiple exons, which undergo alternative splicing. Here, we demonstrate that the human PECAM-1 cytoplasmic domain undergoes alternative splicing, generating six different isoforms. RT-PCR cloning and DNA sequence analysis indicated that human tissue and endothelial cells express multiple isoforms of PECAM-1, including the full-length PECAM-1 and five other isoforms, which lack exon 12, 13, 14, or 15 or exons 14 and 15. The full-length PECAM-1 is the predominant isoform detected in human tissue and endothelial cells. This is in contrast to murine endothelium, in which the PECAM-1 isoform lacking exons 14 and 15 is the predominant isoform. The PECAM-1 isoform lacking exon 13 detected in human tissue and endothelial cells is absent in murine endothelium. The expression pattern of PECAM-1 isoforms changes during tube formation of endothelial cells on Matrigel, which may indicate specialized roles for specific isoforms of PECAM-1 during angiogenesis. The data presented here demonstrate that human PECAM-1 undergoes alternative splicing, generating multiple isoforms in vascular beds of various tissues. Therefore, the regulated expression of these isoforms may influence endothelial cell adhesive properties during angiogenesis and/or vasculogenesis.     

Tissue-specific expression of insulin receptor isoforms in obesity/type 2 diabetes mouse models

The two insulin receptor (IR) isoforms IR-A and IR-B are responsible for the pleiotropic actions of insulin and insulin-like growth factors. Consequently, changes in IR isoform expression and in the bioavailability of their ligands will impact on IR-mediated functions. Although alteration of IR isoform expression has been linked to insulin resistance, knowledge of IR isoform expression and mechanisms underlying tissue/cell-type-specific changes in metabolic disease are lacking. Using mouse models of obesity/diabetes and measuring the mRNA of the IR isoforms and mRNA/protein levels of total IR, we provide a data set of IR isoform expression pattern that documents changes in a tissue-dependent manner. Combining tissue fractionation and a new in situ mRNA hybridization technology to visualize the IR isoforms at cellular resolution, we explored the mechanism underlying the change in IR isoform expression in perigonadal adipose tissue, which is mainly caused by tissue remodelling, rather than by a shift in IR alternative splicing in a particular cell type, e.g. adipocytes.     

Selective Expression of Myosin IC Isoform A in Mouse and Human Cell Lines and Mouse Prostate Cancer Tissues

Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.     

Posttranscriptional regulation of IL-23 expression by IFN-gamma through tristetraprolin

IL-23 plays an essential role in maintenance of IL-17-producing Th17 cells that are involved in the pathogenesis of several autoimmune diseases. Regulation of Th17 cells is tightly controlled by multiple factors such as IL-27 and IFN-γ. However, the detailed mechanisms responsible for IFN-γ-mediated Th17 cell inhibition are still largely unknown. In this study, we demonstrate that IFN-γ differentially regulates IL-12 and IL-23 production in both dendritic cells and macrophages. IFN-γ suppresses IL-23 expression by selectively targeting p19 mRNA stability through its 3'-untranslated region (3'UTR). Furthermore, IFN-γ enhances LPS-induced tristetraprolin (TTP) mRNA expression and protein production. Overexpression of TTP suppresses IL-23 p19 mRNA expression and p19 3'UTR-dependent luciferase activity. Additionally, deletion of TTP completely abolishes IFN-γ-mediated p19 mRNA degradation. We further demonstrate that IFN-γ suppresses LPS-induced p38 phosphorylation, and blockade of p38 MAPK signaling pathway with SB203580 inhibits IFN-γ- and LPS-induced p19 mRNA expression, whereas overexpression of p38 increases p19 mRNA expression via reducing TTP binding to the p19 3'UTR. Finally, inhibition of p38 phosphorylation by IFN-γ leads to TTP dephosphorylation that could result in stronger binding of the TTP to the adenosine/uridine-rich elements in the p19 3'UTR and p19 mRNA degradation. In summary, our results reveal a direct link among TTP, IFN-γ, and IL-23, indicating that IFN-γ-mediated Th17 cell suppression might act through TTP by increasing p19 mRNA degradation and therefore IL-23 inhibition.     

Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules

A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E- cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.     

Vascular circadian rhythms in a mouse vascular smooth muscle cell line (Movas-1)

The circadian system in mammals is a hierarchy of oscillators throughout the organism that are coordinated by the circadian clock in the hypothalamic suprachiasmatic nucleus. Peripheral clocks act to integrate time-of-day information from neural or hormonal signals, regulating gene expression, and, subsequently, organ physiology. However, the mechanisms by which the central clock communicates with peripheral oscillators are not understood and are likely tissue specific. In this study, we establish a mouse vascular cell model suitable for investigations of these mechanisms at a molecular level. Using the immortalized vascular smooth muscle cell line Movas-1, we determined that these cells express the circadian clock machinery with robust rhythms in mRNA expression over a 36-h period after serum shock synchronization. Furthermore, norepinephrine and forskolin were able to synchronize circadian rhythms in bmal1. With synchronization, we observed cycling of specific genes, including the tissue inhibitor of metalloproteinase 1 and 3 (timp1, timp3), collagen 3a1 (col3a1), transgelin 1 (sm22alpha), and calponin 1 (cnn1). Diurnal expression of these genes was also found in vivo in mouse aortic tissue, using microarray and real-time RT-PCR analysis. Both of these revealed ultradian rhythms in genes similar to the cycling observed in Movas-1 in vitro. These findings highlight the cyclical nature of structurally important genes in the vasculature that is similar both in vivo and in vitro. This study establishes the Movas-1 cells as a novel cell model from which to further investigate the molecular mechanisms of clock regulation in the vasculature.     

Molecular cloning of cDNA encoding the rat neural cell adhesion molecule L1. Two L1 isoforms in the cytoplasmic region are produced by differential splicing

We have isolated and sequenced a full-length cDNA encoding the rat neural cell adhesion molecule L1. The deduced amino acid sequence as a whole shows high homology to mouse L1 sequence. In addition to this complete form of L1, we found an isoform, L1cs, which lacks four amino acid residues (RSLE) in the cytoplasmic domain and probably is derived from the same single L1 gene by tissue-specific alternative splicing. While L1 mRNA was predominantly expressed in the brain, L1cs mRNA was found exclusively in peripheral nervous tissue. Differential splicing in the highly conserved cytoplasmic domain may play an important role in modulating the function of L1 in different cells.     

Cytokine signaling through Stat3 activates integrins, promotes adhesion, and induces growth arrest in the myeloid cell line 32D

Hematopoietic cell development and function is dependent on cytokines and on intercellular interactions with the microenvironment. Although the intracellular signaling pathways stimulated by cytokine receptors are well described, little is known about the mechanisms through which these pathways modulate hematopoietic cell adhesion events in the microenvironment. Here we show that cytokine-activated Stat3 stimulates the expression and function of cell surface adhesion molecules in the myeloid progenitor cell line 32D. We generated an erythropoietin receptor (EpoR) isoform (ER343/401-S3) that activates Stat3 rather than Stat5 by substituting the Stat3 binding/activation sequence motif from gp130 for the sequences surrounding tyrosines 343 and 401 in the receptor cytoplasmic region. Activation of Stat3 leads to homotypic cell aggregation, increased expression of intercellular adhesion molecule 1 (ICAM-1), CD18, and CD11b, and activation of signaling through CD18-containing integrins. Unlike the wild type EpoR, ER343/401-S3 is unable to support long term Epo-dependent proliferation in 32D cells. Instead, Epo-treated ER343/401-S3 cells undergo G(1) arrest and express elevated levels of the cyclin-dependent kinase inhibitor p27(Kip1). Sustained activation of Stat3 in these cells is required for their altered morphology and growth properties since constitutive SOCS3 expression abrogates homotypic cell aggregation, signaling through CD18-containing integrins, G(1) arrest, and accumulation of p27(Kip1). Collectively, our results demonstrate that cytokine-activated Stat3 stimulates the expression and function of cell surface adhesion molecules, indicating that a role for Stat3 is to regulate intercellular contacts in myeloid cells.     

Modulation of microtubule dynamics by tau in living cells: implications for development and neurodegeneration

The neural microtubule-associated protein tau binds to and stabilizes microtubules. Because of alternative mRNA splicing, tau is expressed with either 3 or 4 C-terminal repeats. Two observations indicate that differences between these tau isoforms are functionally important. First, the pattern of tau isoform expression is tightly regulated during development. Second, mutation-induced changes in tau RNA splicing cause neuronal cell death and dementia simply by altering the isoform expression ratio. To investigate whether 3- and 4-repeat tau differentially regulate microtubule behavior in cells, we microinjected physiological levels of these two isoforms into EGFP-tubulin-expressing cultured MCF7 cells and measured the effects on the dynamic instability behavior of individual microtubules by time-lapse microscopy. Both isoforms suppressed microtubule dynamics, though to different extents. Specifically, 4-repeat tau reduced the rate and extent of both growing and shortening events. In contrast, 3-repeat tau stabilized most dynamic parameters about threefold less potently than 4-repeat tau and had only a minimal ability to suppress shortening events. These differences provide a mechanistic rationale for the developmental shift in tau isoform expression and are consistent with a loss-of-function model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal cell death and dementia.     

Gene expression during mammalian spermatogenesis. II. Evidence for stage-specific differences in mRNA populations

Gene expression during murine spermatogenesis has been studied using highly enriched populations of cells obtained by velocity sedimentation at unit gravity and further purified by density gradient centrifugation through Percoll. Polypeptides whose synthesis was directed by total cytoplasmic RNA from round spermatids, pachytene spermatocytes, primitive type A spermatogonia, and Sertoli cells in cell-free translation systems have been compared by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. At the level of detection provided by the electrophoretic methods used, each population of cells contained mRNAs encoding over 200 polypeptides, many of which were present in high abundance in all four cell types. However, for each cell type examined, a minimum of 5-10% of these polypeptides appear to be either specific to or greatly enriched within a particular cell type. Analysis of the polysomal and nonpolysomal cell fractions from pachytene spermatocytes and round spermatids revealed that the two compartments share many identical mRNAs but specific mRNAs are selectively compartmentalized between the cell fractions and between the two cell types. Movement between compartments was seen; e.g., some polypeptides encoded by mRNA found primarily in the nonpolysomal fraction of pachytene cells were later seen in the polysomal fraction from round spermatids. Virtually every other combination was also observed. These results suggest that the control of gene expression at the level of selective production of mRNA and selective utilization of mRNA are among the mechanisms involved in regulation of spermatogenic cell differentiation.     

Protein kinase C delta mediates fibroblast growth factor-2-induced interferon-tau expression in bovine trophoblast

Interferon-tau (IFNT) is the trophoblast-secreted factor responsible for establishing and maintaining pregnancy in ruminants. Several uterine- and embryo-derived factors, including fibroblast growth factor-2 (FGF2), regulate IFNT production. The objective of the present study was to decipher the intracellular signaling mechanisms employed by FGF2 to regulate IFNT production. In bovine trophoblast cells (CT1), mitogen-activated protein kinase kinase-dependent pathways mediated constitutive IFNT mRNA concentrations. However, FGF2-mediated increases in IFNT mRNA levels occurs independent of mitogen-activated protein kinase. Exposure to the pan-protein kinase C (PKC) inhibitor, calphostin C, did not affect basal IFNT mRNA levels but limited the ability of FGF2 to increase IFNT mRNA abundance. Also, supplementation with phorbol 12-myristate 13-acetate (PMA) stimulated IFNT mRNA levels to the same extent as with FGF2. PMA and FGF2 cosupplementation did not elicit an additive effect on IFNT mRNA abundance. Pharmacological antagonists for classic PKCs (G?6976) or novel PKCs, including PKC delta (rottlerin), were used to identify the specific PKC isoform utilized by FGF2. Supplementation of CT1 cells with G?6976 did not affect FGF2 or PMA activities, whereas rottlerin prevented FGF2- and PMA-dependent increases in IFNT mRNA abundance in CT1 cells. Rottlerin also prevented FGF2 from increasing IFNT mRNA levels in Vivot trophoblast cells and primary trophoblast outgrowths. Modifications in PRKCD phosphorylation status were evident following FGF2 and PMA treatment. Also, reducing PRKCD expression by RNA interference attenuated FGF2-dependent increases in IFNT mRNA abundance. In conclusion, these results provide evidence that FGF2 regulates IFNT production in bovine trophectoderm by acting through PRKCD.