Increased expression level of the splicing variant of SIP1 in motor neuron diseases

Survival motor neuron (SMN) interacting protein 1 (SIP1) interacts with SMN protein and plays a crucial role in the biogenesis of spliceosomes. We have identified three novel splicing variants of the SIP1 (SIP1-beta, -gamma and -delta), in addition to the full-length SIP1-alpha. SIP1-alpha as found to be ubiquitously expressed at high levels in the various normal tissues examined. In contrast, SIP1-beta and -gamma were expressed at very low levels in these tissues. In muscle specimens from patients with spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), the expression of SIP1-alpha was dramatically decreased compared to that observed in the normal tissues. In addition, the expression of SIP1-beta was significantly increased in tissues derived from patients with either disease. These findings suggest that an aberrant alternative splicing event in SIP1 occurs tissues derived from patients with the motor neuron diseases, and contributes to the pathological process of SMA and ALS.     

Murine p38-delta mitogen-activated protein kinase, a developmentally regulated protein kinase that is activated by stress and proinflammatory cytokines

The p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression. We have isolated the murine cDNA clones encoding p38-delta MAPK, and we have localized the p38-delta gene to mouse chromosome 17A3-B and human chromosome 6p21.3. By using Northern and in situ hybridization, we have examined the expression of p38-delta in the mouse adult tissues and embryos. p38-delta was expressed primarily in the lung, testis, kidney, and gut epithelium in the adult tissues. Although p38-delta was expressed predominantly in the developing gut and the septum transversum in the mouse embryo at 9.5 days, its expression began to be expanded to many specific tissues in the 12.5-day embryo. At 15.5 days, p38-delta was expressed virtually in most developing epithelia in embryos, suggesting that p38-delta is a developmentally regulated MAPK. Interestingly, p38-delta and p38-alpha were similar serine/threonine kinases but differed in substrate specificity. Overall, p38-delta resembles p38-gamma, whereas p38-beta resembles p38-alpha. Moreover, p38-delta is activated by environmental stress, extracellular stimulants, and MAPK kinase-3, -4, -6, and -7, suggesting that p38-delta is a unique stress-responsive protein kinase.     

Characterization of the structure and transcriptional patterns of the gene encoding the late histone subtype H1-beta of the sea urchin Strongylocentrotus purpuratus.

We have cloned and characterized the gene encoding the late histone H1-beta subtype from the sea urchin Strongylocentrotus purpuratus. The gene contains all of the upstream sequence homologies previously seen in late H1-gamma genes. The expression of H1-beta mRNA is coordinated with that of H1-gamma mRNA, and like H1-gamma it is expressed in all adult somatic tissues tested.     

PKC-zeta expression is lower in osteoblasts from arthritic patients: IL1-beta and TNF-alpha induce a similar decrease in non-arthritic human osteoblasts

Protein kinase C (PKC) is a family of enzymes detected in a diverse range of cell types where they regulate various cellular functions such as proliferation, differentiation, cytoskeletal remodelling, cytokine production, and receptor-mediated signal transduction. In this study we have analyzed the expression of 11 PKC isoforms (-alpha, -beta(I), -beta(II), -gamma, -delta, -eta, -theta, -epsilon, -zeta, -iota/lambda, and -micro) in osteoblasts from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) in comparison with osteoblasts from post-traumatic (PT) patients. By Western blotting analysis, nine isoforms, -alpha, -beta(I), -beta(II), -delta, -theta, - epsilon, -zeta, - iota/lambda, and -micro, were detected in osteoblasts. In RA and OA patients, PKC -theta and -micro were greater expressed whereas PKC-epsilon and -zeta decreased when compared with normal cells. The subcellular distribution and quantitative differences were confirmed by immuno-electron microscopy. Furthermore, we demonstrated that treatment with the proinflammatory cytokines, IL-1beta and TNF-alpha, significantly decreased PKC-zeta expression in PT osteoblasts. This suggests that proinflammatory cytokines can modulate the expression of this PKC isoform in osteoblasts in a way which is similar to changes detected in arthritic patients.     

The mouse keratin 6 isoforms are differentially expressed in the hair follicle, footpad, tongue and activated epidermis

Keratin 6 (K6) is expressed constitutively in a variety of internal stratified epithelia as well as in palmoplantar epidermis and in specialized cells of the hair follicle. K6 expression can also be induced by hyperproliferative conditions as in wound healing or by conditions that perturb normal keratinocyte function. The functional significance of the expression of K6 on keratinocyte biology under these disparate conditions is not known. Here we report on the characterization of two isoforms of mouse K6 that are encoded by separate genes. The two genes (denoted K6a and K6b) are linked, have the same orientation and are actively transcribed. Sequence analysis revealed, that although they encode almost identical products, they have distinctly different regulatory regions, suggesting that the two K6 genes would be differentially expressed. In an attempt to define the expression characteristics of the K6 isoforms, we produced transgenic mice with each gene after modifying the C-terminal sequences to enable detection of the transgenic proteins with specific antibodies. The constitutive expression of the K6a transgene paralleled that of the endogenous genes in all K6 expressing tissues, except in the tongue. The K6b transgene was also expressed in these tissues but, in contrast to K6a, was only expressed in suprabasal cells. Both K6 transgenes were also induced in the interfollicular epidermis in response to phorbol esters, with K6a induced in all layers of the treated epidermis, while K6b was expressed only in suprabasal cells. These studies suggest that the K6 isoforms have overlapping yet distinct expression profiles.     

Suprabasal desmoglein 3 expression in the epidermis of transgenic mice results in hyperproliferation and abnormal differentiation

The desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) isoforms of the desmosomal cadherins are expressed in the suprabasal layers of epidermis, whereas Dsg3 and Dsc3 are more strongly expressed basally. This differential expression may have a function in epidermal morphogenesis and/or may regulate the proliferation and differentiation of keratinocytes. To test this hypothesis, we changed the expression pattern by overexpressing human Dsg3 under the control of the keratin 1 (K1) promoter in the suprabasal epidermis of transgenic mice. From around 12 weeks of age, the mice exhibited flaking of the skin accompanied by epidermal pustules and thinning of the hair. Histological analysis of affected areas revealed acanthosis, hypergranulosis, hyperkeratosis, localized parakeratosis, and abnormal hair follicles. This phenotype has some features in common with human ichthyosiform diseases. Electron microscopy revealed a mild epidermal spongiosis. Suprabasally, desmosomes showed incorporation of the exogenous protein by immunogold labeling but were normal in structure. The epidermis was hyperproliferative, and differentiation was abnormal, demonstrated by expression of K14 in the suprabasal layer, restriction of K1, and strong induction of K6 and K16. The changes resembled those found in previous studies in which growth factors, cytokines, and integrins had been overexpressed in epidermis. Thus our data strongly support the view that Dsg3 contributes to the regulation of epidermal differentiation. Our results contrast markedly with those recently obtained by expressing Dsg3 in epidermis under the involucrin promoter. Possible reasons for this difference are considered in this paper.     

The mouse dead-end gene isoform alpha is necessary for germ cell and embryonic viability

Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform alpha (DND1-alpha) and DND1-isoform beta (DND1-beta). Using isoform-specific antibodies, we determined DND1-alpha is expressed in embryos and embryonic gonads whereas DND1-beta expression is restricted to germ cells of the adult testis. Our data implicate DND1-alpha isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain.     

The lanceolate hair rat phenotype results from a missense mutation in a calcium coordinating site of the desmoglein 4 gene

Desmosomal cadherins are essential cell adhesion molecules present throughout the epidermis and other organs, whose major function is to provide mechanical integrity and stability to epithelial cells in a wide variety of tissues. We recently identified a novel desmoglein family member, Desmoglein 4 (Dsg4), using a positional cloning approach in two families with localized autosomal recessive hypotrichosis (LAH) and in the lanceolate hair (lah) mouse. In this study, we report cloning and identification of the rat Dsg4 gene, in which we discovered a missense mutation in a naturally occurring lanceolate hair (lah) rat mutant. Phenotypic analysis of lah/lah mutant rats revealed a striking hair shaft defect with the appearance of a lance head within defective hair shafts. The mutation disrupts a critical calcium binding site bridging the second and third extracellular domains of Dsg4, likely disrupting extracellular interactions of the protein.     

Inhibitory effect of interferon-beta on mouse spleen-derived mast cells

Preparations of murine recombinant interferon (Mu-rIFN)-alpha, -beta and -gamma were assessed for their influence on in vitro growth of mast cells from normal mouse spleen cells (Sp C). Mast cell growth was inhibited by Mu-rIFNs when Sp C were exposed throughout the entire culture period to Mu-rIFNs. The most potent inhibitor of mast cell growth was Mu-rIFN-gamma, followed by Mu-rIFN-beta; Mu-rlFN-alpha had little effect. When added to IC-2 cells, clonal mast cell progenitor, both Mu-rlFN-beta and -gamma), significantly inhibited proliferative response of the target cells. The suppressive effect of Mu-rIFNs on IC-2 cells was selectively abolished by monoclonal antibodies against Mu-rIFN-beta and -gamma.     

Desmoglein 4 is expressed in highly differentiated keratinocytes and trichocytes in human epidermis and hair follicle

Desmosomes are critical for the tissue integrity of stratified epithelia and their appendages. Desmogleins (DSGs) and desmocollins (DSCs) are transmembrane desmosomal cadherins that interact extracellularly to link neighboring epithelial cells. We recently identified a new member of the DSG family, designated desmoglein 4, whose mutations cause hypotrichosis in human, mouse and rat. In this study, we analyzed in detail the expression domains of human desmoglein 4 protein (DSG4) in human skin relative to differentiation markers and other DSGs. Our results show that DSG4 protein is expressed in the more highly differentiated layers of the epidermis. This expression pattern in vivo is recapitulated in highly differentiated HaCaT human keratinocytes and normal human keratinocytes in vitro. In the human hair follicle, DSG4 is expressed specifically in the hair shaft cortex, the lower hair cuticle, and the upper inner root sheath (IRS) cuticle. Using a green fluorescent protein-tagged version of mouse or rat desmoglein 4 protein (Dsg4) and immuno-electron microscopy, we demonstrate that Dsg4 localizes to desmosomes both in vitro and in vivo. The highly specific expression pattern of DSG4 in the human hair follicle, combined with the phenotype of rodent models and human patients with desmoglein 4 mutations, underscores the importance of this adhesion molecule in the integrity of the hair shaft.     

Nesprins: Tissue-Specific Expression of Epsilon and Other Short Isoforms

Nesprin-1-giant and nesprin-2-giant regulate nuclear positioning by the interaction of their C-terminal KASH domains with nuclear membrane SUN proteins and their N-terminal calponin-homology domains with cytoskeletal actin. A number of short isoforms lacking the actin-binding domains are produced by internal promotion. We have evaluated the significance of these shorter isoforms using quantitative RT-PCR and western blotting with site-specific monoclonal antibodies. Within a complete map of nesprin isoforms, we describe two novel nesprin-2 epsilon isoforms for the first time. Epsilon isoforms are similar in size and structure to nesprin-1-alpha. Expression of nesprin isoforms was highly tissue-dependent. Nesprin-2-epsilon-1 was found in early embryonic cells, while nesprin-2-epsilon-2 was present in heart and other adult tissues, but not skeletal muscle. Some cell lines lack shorter isoforms and express only one of the two nesprin genes, suggesting that either of the giant nesprins is sufficient for basic cell functions. For the first time, localisation of endogenous nesprin away from the nuclear membrane was shown in cells where removal of the KASH domain by alternative splicing occurs. By distinguishing between degradation products and true isoforms on western blots, it was found that previously-described beta and gamma isoforms are expressed either at only low levels or with a limited tissue distribution. Two of the shortest alpha isoforms, nesprin-1-alpha-2 and nesprin-2-alpha-1, were found almost exclusively in cardiac and skeletal muscle and a highly conserved and alternatively-spliced exon, available in both nesprin genes, was always included in these tissues. These “muscle-specific” isoforms are thought to form a complex with emerin and lamin A/C at the inner nuclear membrane and mutations in all three proteins cause Emery-Dreifuss muscular dystrophy and/or inherited dilated cardiomyopathy, disorders in which only skeletal muscle and/or heart are affected.