SUMMARY Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss Muscular Dystrophy (AD-EDMD) and Hutchinson-Gilford Progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna−/−) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna−/− and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna−/− or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 over-accumulation in LMNA mutant fibroblasts and in HGPS cells corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna−/−, LmnaΔ9, and HGPS disorders.

Mutations in the key enzyme of sialic acid biosynthesis, uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK), result in hereditary inclusion body myopathy (HIBM), an adult-onset, progressive neuromuscular disorder. We created knockin mice harboring the M712T Gne/Mnk mutation. Homozygous mutant (Gne M712T/M712T ) mice did not survive beyond P3. At P2, significantly decreased Gne-epimerase activity was observed in Gne M712T/M712T muscle, but no myopathic features were apparent. Rather, homozygous mutant mice had glomerular hematuria, proteinuria, and podocytopathy. Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration yielded survival beyond P3 in 43% of the Gne M712T/M712T pups. Survivors exhibited improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gneepimerase activities. These findings establish this Gne M712T/M712T knockin mouse as what we believe to be the first genetic model of podocyte injury and segmental glomerular basement membrane splitting due to hyposialylation. The results also support evaluation of ManNAc as a treatment not only for HIBM but also for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane.

Nuclear factor kappa B (NF‐κB) is a key mediator of inflammation. Unchecked NF‐κB signalling can engender autoimmune pathologies and cancers. Here, we show that Tax1‐binding protein 1 (TAX1BP1) is a negative regulator of TNF‐α‐ and IL‐1β‐induced NF‐κB activation and that binding to mono‐ and polyubiquitin by a ubiquitin‐binding Zn finger domain in TAX1BP1 is needed for TRAF6 association and NF‐κB inhibition. Mice genetically knocked out for TAX1BP1 are born normal, but develop age‐dependent inflammatory cardiac valvulitis, die prematurely, and are hypersensitive to low doses of TNF‐α and IL‐1β. TAX1BP1−/− cells are more highly activated for NF‐κB than control cells when stimulated with TNF‐α or IL‐1β. Mechanistically, TAX1BP1 acts in NF‐κB signalling as an essential adaptor between A20 and its targets.

The S1P 2 receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite sphingosine 1-phosphate with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S1P 2 receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S1P 2 receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S1P 2 receptor-null mice showed markedly disturbed structures. JTE013, an S1P 2 receptor-specific antagonist, blocked the S1P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S1P 2 receptor-null mice. Sphingosine 1-phosphate (S1P)3 is a sphingolipid metabolite that functions as a signaling ligand through interactions with G protein-coupled S1P receptors. Five high affinity receptors (S1P 1 -S1P 5 ) have been described that trigger distinctive intracellular signaling pathways (1, 2) following binding of the S1P ligand. Three of these receptors, S1P 1 , S1P 2 , and S1P 3 , are widely expressed on cells and tissues, whereas expression of the S1P 4 and S1P 5 receptors are largely confined to cells of the immune and nervous systems. The ligand S1P is produced through the phosphorylation of sphingosine by sphingosine kinases 1 and 2 and can be degraded by S1P-specific enzymes that include phosphatases and a lyase (3). Micromolar levels of the S1P ligand, bound primarily to high density lipoproteins, are found in plasma and may provide a source for tonic signaling. Acute S1P signaling may result from enhanced secretion of S1P from cells, such as platelets and mast cells, upon activation.Genetic deletion of receptors within mice has been an important means of identifying the physiologic roles of S1P receptor signaling. These studies have demonstrated that the signaling pathways are biologically significant and potentially clinically relevant within the vascular (4 -6), immune (7, 8), pulmonary (9), and nervous systems (10).Here we report that S1P 2 receptor expression is essential for proper functioning of the auditory and vestibular systems. S1P 2 receptor-null mice exhibit profound deafness early in life with severe associated pathologic changes within the cochlea. Early cellular defects were found to be in the stria vascularis, a compartment that harbors the main vasculature of the inner ea...

Acute lymphoblastic leukemia (ALL) is a clonal disease that evolves through the accrual of genetic rearrangements and͞or mutations within the dominant clone. The TEL-AML1 (ETV6-RUNX1) fusion in precursor-B (pre-B) ALL is the most common genetic rearrangement in childhood cancer; however, the cellular origin and the molecular pathogenesis of TEL-AML1-induced leukemia have not been identified. To study the origin of TEL-AML1-induced ALL, we generated transgenic zebrafish expressing TEL-AML1 either ubiquitously or in lymphoid progenitors. TEL-AML1 expression in all lineages, but not lymphoid-restricted expression, led to progenitor cell expansion that evolved into oligoclonal B-lineage ALL in 3% of the transgenic zebrafish. This leukemia was transplantable to conditioned wildtype recipients. We demonstrate that TEL-AML1 induces a B cell differentiation arrest, and that leukemia development is associated with loss of TEL expression and elevated Bcl2͞Bax ratio. The TEL-AML1 transgenic zebrafish models human pre-B ALL, identifies the molecular pathways associated with leukemia development, and serves as the foundation for subsequent genetic screens to identify modifiers and leukemia therapeutic targets.stem cell ͉ translocation ͉ childhood cancer ͉ genetics T he TEL-AML1 fusion generated by the t(12, 21)(p13;q22) chromosomal translocation is present in 25% of childhood pre-B acute lymphoblastic leukemia (ALL), making it the most common genetic rearrangement in childhood cancer (1-3). The translocation fuses the first five exons of the Ets transcription factor TEL (also known as ETV6) in-frame to nearly the entire AML1 gene (also known as RUNX1). Retrospective studies in twins with pre-B ALL, as well as Guthrie cards studies from 567 normal newborns (4), reveal that the TEL-AML1 fusion occurs in utero, with a protracted time course for leukemia development (5, 6).Murine studies involving TEL-AML1 suggest that this fusion protein confers a low transforming ability. Transgenic mice expressing TEL-AML1 from the Ig heavy chain promoter (E) did not develop any hematological disorder (7). Mice transplanted with bone marrow cells transduced with retroviral vectors expressing TEL-AML1 developed a preleukemic state without occult leukemia (8-10). The incidence of leukemia in such mice increased only in the presence of cooperating mutations (11).The cell initially transformed by TEL-AML1 remains to be elucidated; however, in ALL patients, the TEL-AML1 fusion event precedes differentiation of lymphoid progenitors to pre-B cells (12). This finding confines the origin of pre-B ALL to a B-lineage restricted progenitor(s) (4) or a multipotent hematopoietic stem cell (HSC) with preferential B-lymphoid clonal expansion (13).We used the zebrafish to study TEL-AML1 leukemogenesis for several reasons. First, the zebrafish has well conserved genetic processes controlling hematopoesis (14, 15). Second, zebrafish develop tumors that are histologically similar to human tumors (16)(17)(18)(19)(20). The lymphoid expression of mouse c-Myc led to...

Here we describe and characterize a small serine/threonine kinase (SSTK) which consists solely of the Nand C-lobes of a protein kinase catalytic domain. SSTK protein is highly conserved among mammals, and no close homologues were found in the genomes of nonmammalian organisms. SSTK specifically interacts with HSP90-1␤, HSC70, and HSP70 proteins, and this association appears to be required for SSTK kinase activity. The SSTK transcript was most abundant in human and mouse testes but was also detected in all human tissues tested. In the mouse testis, SSTK protein was localized to the heads of elongating spermatids. Targeted deletion of the SSTK gene in mice resulted in male sterility due to profound impairment in motility and morphology of spermatozoa. A defect in DNA condensation in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins. SSTK phosphorylated histones H1, H2A, H2AX, and H3 but not H2B or H4 or transition protein 1 in vitro. These results demonstrate that SSTK is required for proper postmeiotic chromatin remodeling and male fertility. Abnormal sperm chromatin condensation is common in sterile men, and our results may provide insight into the molecular mechanisms underlying certain human infertility disorders.Phosphorylation of serine, threonine, and tyrosine residues in substrate targets by protein kinases is a common posttranslational protein modification in eukaryotes and provides a fundamental mechanism for the control of cellular events. Cell division and growth, adhesion and migration, metabolic activity and responses upon environmental stimuli, cell to cell communication, signal transduction, and apoptosis are among the many processes regulated by protein kinases (15,16). At the molecular level, phosphorylation and dephosphorylation of enzymes allow fast and sensitive regulation of enzyme activity and are also a major mechanism of transmembrane signaling and signal amplification in the branching network of intracellular protein kinase cascades that ultimately control gene expression by phosphorylation of transcription factors. Phosphorylation of protein substrates can provide binding sites for protein domains which recognize specific phosphorylated amino acid sequences, thereby mediating protein-protein interactions. Protein kinases constitute a large superfamily of related enzymes which contain 12 conserved subdomains folded into N-and C-lobes of the catalytic domain. The superfamily is subdivided into protein serine/threonine kinases, protein tyrosine kinases, and atypical kinases on the basis of substrate specificity (14,16).Phosphorylation events play a central role in chromatin remodeling during mitosis in somatic cells and during meiosis in mammalian spermiogenesis (2, 10). Much less is known about the mechanisms of postmeiotic chromatin condensation. The structure of chromatin changes dramatically during postmeiotic spermiogenesis, as germ cells develop from round spermatids to fully differ...

A gene causing autosomal-recessive, nonsyndromic hearing loss, DFNB39, was previously mapped to an 18 Mb interval on chromosome 7q11.22-q21.12. We mapped an additional 40 consanguineous families segregating nonsyndromic hearing loss to the DFNB39 locus and refined the obligate interval to 1.2 Mb. The coding regions of all genes in this interval were sequenced, and no missense, nonsense, or frameshift mutations were found. We sequenced the noncoding sequences of genes, as well as noncoding genes, and found three mutations clustered in intron 4 and exon 5 in the hepatocyte growth factor gene (HGF). Two intron 4 deletions occur in a highly conserved sequence that is part of the 3' untranslated region of a previously undescribed short isoform of HGF. The third mutation is a silent substitution, and we demonstrate that it affects splicing in vitro. HGF is involved in a wide variety of signaling pathways in many different tissues, yet these putative regulatory mutations cause a surprisingly specific phenotype, which is nonsydromic hearing loss. Two mouse models of Hgf dysregulation, one in which an Hgf transgene is ubiquitously overexpressed and the other a conditional knockout that deletes Hgf from a limited number of tissues, including the cochlea, result in deafness. Overexpression of HGF is associated with progressive degeneration of outer hair cells in the cochlea, whereas cochlear deletion of Hgf is associated with more general dysplasia.

MicroRNAs (miRNAs) act in post-transcriptional gene silencing and are proposed to function in a wide spectrum of pathologies, including cancers and viral diseases. Currently, to our knowledge, no detailed mechanistic characterization of small molecules that interrupt miRNA pathways have been reported. In screening a small chemical library, we identified compounds that suppress RNA interference activity in cultured cells. Two compounds were characterized; one impaired Dicer activity while the other blocked small RNA-loading into an Argonaute 2 (AGO2) complex. We developed a cell-based model of miRNAdependent tumorigenesis, and using this model, we observed that treatment of cells with either of the two compounds effectively neutralized tumor growth. These findings indicate that miRNA pathway-suppressing small molecules could potentially reverse tumorigenesis.