Natural inhibitor of transforming growth factor-beta protects against scarring in experimental kidney disease.

The central pathological feature of human kidney disease that leads to kidney failure is the accumulation of extracellular matrix in glomeruli. Overexpression of transforming growth factor-beta (TGF-beta) underlies the accumulation of pathological matrix in experimental glomerulonephritis. Administration of an antibody raised against TGF-beta to glomerulonephritic rats suppresses glomerular matrix production and prevents matrix accumulation in the injured glomeruli. One of the matrix components induced by TGF-beta, the proteoglycan decorin, can bind TGF-beta and neutralize its biological activity, so decorin may be a natural regulator of TGF-beta (refs 3, 4). We tested whether decorin could antagonize the action of TGF-beta in vivo using the experimental glomerulonephritis model. We report here that administration of decorin inhibits the increased production of extracellular matrix and attenuates manifestations of disease, confirming our hypothesis. On the basis of our results, decorin may eventually prove to be clinically useful in diseases associated with overproduction of TGF-beta.     

Deactivation of macrophages by transforming growth factor-beta

Macrophage activation--enhanced capacity to kill, in a cell that otherwise mostly scavenges--is essential for host survival from infection and contributes to containment of tumours. Both microbes and tumour cells, therefore, may be under pressure to inhibit or reverse the activation of macrophages. This reasoning led to the demonstration of macrophage deactivating factors from both microbes and tumour cells. In some circumstances the host itself probably requires the ability to deactivate macrophages. Macrophages are essential to the healing of wounds and repair of tissues damaged by inflammation. Yet the cytotoxic products of the activated macrophages can damage endothelium, fibroblasts, smooth muscle and parenchymal cells (reviewed in ref. 6). Thus, after an inflammatory site has been sterilized, the impact of macrophage activation on the host might shift from benefit to detriment. These concepts led us to search for macrophage deactivating effects among polypeptide growth factors that regulate angiogenesis, fibrogenesis and other aspects of tissue repair. Among 11 such factors, two proteins that are 71% similar proved to be potent macrophage deactivators: these are transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2.     

Animal model of Gaucher's disease from targeted disruption of the mouse glucocerebrosidase gene.

Gaucher's disease is the most prevalent lysosomal storage disorder in humans and results from an autosomally inherited deficiency of the enzyme glucocerebrosidase (beta-D-glucosyl-N-acylsphingosine glucohydrolase), which is responsible for degrading the sphingolipid glucocerebroside. An animal model for Gaucher's disease would be important for investigating its phenotypic diversity and pathogenesis and for evaluating therapeutic approaches. A naturally occurring canine model has been reported but not propagated. Attempts to mimic the disease in animals by inhibiting glucocerebrosidase have been inadequate. Here we generate an animal model for Gaucher's disease by creating a null allele in embryonic stem cells through gene targeting and using these genetically modified cells to establish a mouse strain carrying the mutation. Mice homozygous for this mutation have less than 4% of normal glucocerebrosidase activity, die within twenty-four hours of birth and store glucocerebroside in lysosomes of cells of the reticuloendothelial system.     

Negative regulation of transforming growth factor-beta by the proteoglycan decorin

Decorin is a small chondroitin-dermatan sulphate proteoglycan consisting of a core protein and a single glycosaminoglycan chain. Eighty per cent of the core protein consists of 10 repeats of a leucin-rich sequence of 24 amino acids. Similar repeats have been found in two other proteoglycans, biglycan and fibromodulin, and in several other proteins including Drosophila morphogenetic proteins. Expression of high levels of decorin in Chinese hamster ovary cells has a dramatic effect on their morphology and growth properties. We now report that this effect is due at least in part to the ability of decorin to bind transforming growth factor-beta, an autocrine factor that stimulates the growth of Chinese hamster ovary cells. As transforming growth factor-beta induces synthesis of decorin in many cell types, our results suggest that decorin may be a component of a feedback system regulating cell growth.     

Two forms of transforming growth factor-beta distinguished by multipotential haematopoietic progenitor cells

Type-beta transforming growth factors (TGF-beta s) are polypeptides that act hormonally to control proliferation and differentiation of many cell types. Two distinct homodimeric TGF-beta polypeptides, TGF-beta 1 and TGF-beta 2 have been identified which show approximately 70% amino-acid sequence similarity. Despite their structural differences, TGF-beta 1 and TGF-beta 2 are equally potent at inhibiting epithelial cell proliferation and adipogenic differentiation. The recent immunohistochemical localization of high levels of TGF-beta in the bone marrow and haematopoietic progenitors of the fetal liver has raised the possibility that TGF-beta s might be involved in the regulation of haematopoiesis. Here we show that TGF-beta 1, but not TGF-beta 2, is a potent inhibitor of haematopoietic progenitor cell proliferation. TGF-beta 1 inhibited colony formation by murine factor-dependent haematopoietic progenitor cells in response to interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF), as well as colony formation by marrow progenitor cells responding to CSF-1 (M-CSF). The progenitor cell lines examined were approximately 100-fold more sensitive to TGF-beta 1 than TGF-beta 2, and displayed type-I TGF-beta receptors with affinity approximately 20-fold higher for TGF-beta 1 than TGF-beta 2. These results identify TGF-beta 1 as a novel regulator of haematopoiesis that acts through type-I TGF-beta receptors to modulate proliferation of progenitor cells in response to haematopoietic growth factors.     

Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells

The partial amino-acid sequence of purified human transforming growth factor-beta (TGF-beta) was used to identify a series of cDNA clones encoding the protein. The cDNA sequence indicates that the 112-amino acid monomeric form of the natural TGF-beta homodimer is derived proteolytically from a much longer precursor polypeptide which may be secreted. TGF-beta messenger RNA is synthesized in various normal and transformed cells.     

An unusual feature revealed by the crystal structure at 2.2 A resolution of human transforming growth factor-beta 2.

Transforming growth factor type beta 2 (TGF-beta 2) is a member of an expanding family of growth factors that regulate proliferation and differentiation of many different cell types. TGF-beta 2 binds to various receptors, one of which was shown to be a serine/threonine kinase. TGF-beta 2 is involved in wound healing, bone formation and modulation of immune functions. We report here the crystal structure of TGF-beta 2 at 2.2 A resolution, which reveals a novel monomer fold and dimer association. The monomer consists of two antiparallel pairs of beta-strands forming a flat curved surface and a separate, long alpha-helix. The disulphide-rich core has one disulphide bone pointing through a ring formed by the sequence motifs Cys-Ala-Gly-Ala-Cys and Cys-Lys-Cys, which are themselves connected through the cysteines. Two monomers are connected through a single disulphide bridge and associate such that the helix of one subunit interacts with the concave beta-sheet surface of the other. Four exposed loop regions might determine receptor specificity. The structure provides a suitable model for the TGF-beta s and other members of the super-family and is the basis for the analysis of the TGF-beta 2 interactions with the receptor.     

Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene hox-1.5.

Gene targeting in mouse embryo-derived stem cells has been used to disrupt the homeobox gene hox-1.5. Mice heterozygous at the hox-1.5 locus appear normal, whereas hox-1.5-/hox-1.5- mice die at or shortly after birth. These homozygotes are athymic, aparathyroid, have reduced thyroid and submaxillary tissue and exhibit a wide range of throat abnormalities. In addition, they often feature defects of the heart and arteries as well as craniofacial abnormalities. These deficiencies are remarkably similar to the pathology of the human congenital disorder DiGeorge's syndrome.     

Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development

The int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signalling. We have now explored the function of int-1 in the mouse by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis revealed severe abnormalities in the development of the mesencephalon and metencephalon indicating a prominent role for the int-1 protein is in the induction of the mesencephalon and cerebellum.     

Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus

The complete nucleotide sequence of the transforming gene of a mouse sarcoma virus has been determined. It codes for a protein of 374 amino acids. The nucleotide sequence of the junctions between a murine leukaemia virus and cellular sequences leading to the formation of the viral transforming gene have also been elucidated. The viral transforming sequence and its cellular homologue share an uninterrupted stretch of 1,159 nucleotides, with few base substitutions. The predicted amino acid sequence of the mouse sarcoma virus transforming gene was found to share considerable homology with the proposed amino acid sequence of the avian sarcoma virus oncogene (src) product.     

Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-beta

Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hormone (FSH), of gonadal origin and thus a potential contraceptive, may constitute a missing link in the mechanism controlling the differential secretion of the pituitary gonadotropins. Inhibin-like bioactivity has been reported in various fluids and extracts of testis and in ovarian follicular fluid. Although there have been several attempts to purify inhibin from seminal plasma, purification from follicular fluid has been more successful (refs 14-16; for review see ref. 17). We have previously isolated two forms (A and B) of inhibin from porcine follicular fluid. Each form comprised two dissimilar subunits of relative molecular mass (Mr) 18,000 (18K, referred to here as the alpha-subunit) and 14K (the beta-subunit), crosslinked by one or more disulphide bridge(s). Forms A and B differ in the N-terminal sequence of their 14K subunit. Preliminary structural characterization of porcine and bovine ovarian inhibins shows that they have similar properties. Here, we have used the N-terminal amino-acid sequence data on the subunits of each inhibin to identify cloned complementary DNAs encoding the biosynthetic precursors and report that inhibins are the product of a gene family that also includes transforming growth factor-beta (TGF-beta) and whose structural organization is similar to that of pituitary and placental glycoprotein hormones.     

Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion.

Burnet's clonal selection theory suggests that each B lymphocyte is committed to a single antibody specificity. This is achieved by a programme of somatic rearrangements of the gene segments encoding antibody variable (V) regions, in the course of B-cell development. Evidence from immunoglobulin-transgenic mice and immunoglobulin-gene-transfected transformed pre-B cells suggest that the membrane form of the immunoglobulin heavy (H) chain of class mu (microns), expressed from a rearranged H-chain (IgH) locus, may signal allelic exclusion of the homologous IgH locus in the cell and initiation of light (L)-chain gene rearrangement in the Ig kappa loci. We report here that targeted disruption of the membrane exon of the mu chain indeed results in the loss of H-chain allelic exclusion. But, some kappa chain gene rearrangement is still observed in the absence of micron expression.     

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.     

ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent's disease

Dent's disease is an X-linked disorder associated with the urinary loss of low-molecular-weight proteins, phosphate and calcium, which often leads to kidney stones. It is caused by mutations in ClC-5, a renal chloride channel that is expressed in endosomes of the proximal tubule. Here we show that disruption of the mouse clcn5 gene causes proteinuria by strongly reducing apical proximal tubular endocytosis. Both receptor-mediated and fluid-phase endocytosis are affected, and the internalization of the apical transporters NaPi-2 and NHE3 is slowed. At steady state, however, both proteins are redistributed from the plasma membrane to intracellular vesicles. This may be caused by an increased stimulation of luminal parathyroid hormone (PTH) receptors owing to the observed decreased tubular endocytosis of PTH. The rise in luminal PTH concentration should also stimulate the hydroxylation of 25(OH) vitamin D3 to the active hormone. However, this is counteracted by a urinary loss of the precursor 25(OH) vitamin D3. The balance between these opposing effects, both of which are secondary to the defect in proximal tubular endocytosis, probably determines whether there will be hypercalciuria and kidney stones.     

Developmental defects of the ear, cranial nerves and hindbrain resulting from targeted disruption of the mouse homeobox gene Hox-1.6.

Gene targeting in mouse embryo-derived stem cells has been used to generate mice with a disruption in the homeobox gene Hox-1.6. Mice heterozygous at the Hox-1.6 locus appear normal, whereas Hox-1.6-/Hox-1.6- mice die at or shortly after birth. These homozygotes exhibit profound defects in the formation of the external, middle and inner ears as well as in specific hindbrain nuclei, and in cranial nerves and ganglia. The affected tissues lie within a narrow region along the anteroposterior axis of the mouse but are of diverse embryonic origin. The set of defects associated with the disruption of Hox-1.6 is distinct from and nonoverlapping with that of the closely linked Hox-1.5 gene. But both mutations cause loss, rather than homeotic transformation, of tissues and structures.     

Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease.

Huntington's disease is an autosomal-dominant progressive neurodegenerative disorder resulting in specific neuronal loss and dysfunction in the striatum and cortex. The disease is universally fatal, with a mean survival following onset of 15-20 years and, at present, there is no effective treatment. The mutation in patients with Huntington's disease is an expanded CAG/polyglutamine repeat in huntingtin, a protein of unknown function with a relative molecular mass of 350,000 (M(r) 350K). The length of the CAG/polyglutamine repeat is inversely correlated with the age of disease onset. The molecular pathways mediating the neuropathology of Huntington's disease are poorly understood. Transgenic mice expressing exon 1 of the human huntingtin gene with an expanded CAG/polyglutamine repeat develop a progressive syndrome with many of the characteristics of human Huntington's disease. Here we demonstrate evidence of caspase-1 activation in the brains of mice and humans with the disease. In this transgenic mouse model of Huntington's disease, expression of a dominant-negative caspase-1 mutant extends survival and delays the appearance of neuronal inclusions, neurotransmitter receptor alterations and onset of symptoms, indicating that caspase-1 is important in the pathogenesis of the disease. In addition, we demonstrate that intracerebroventricular administration of a caspase inhibitor delays disease progression and mortality in the mouse model of Huntington's disease.     

Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption

Aquaporin-1 (AQP1) is a water channel protein expressed widely in vascular endothelia, where it increases cell membrane water permeability. The role of AQP1 in endothelial cell function is unknown. Here we show remarkably impaired tumour growth in AQP1-null mice after subcutaneous or intracranial tumour cell implantation, with reduced tumour vascularity and extensive necrosis. A new mechanism for the impaired angiogenesis was established from cell culture studies. Although adhesion and proliferation were similar in primary cultures of aortic endothelia from wild-type and from AQP1-null mice, cell migration was greatly impaired in AQP1-deficient cells, with abnormal vessel formation in vitro. Stable transfection of non-endothelial cells with AQP1 or with a structurally different water-selective transporter (AQP4) accelerated cell migration and wound healing in vitro. Motile AQP1-expressing cells had prominent membrane ruffles at the leading edge with polarization of AQP1 protein to lamellipodia, where rapid water fluxes occur. Our findings support a fundamental role of water channels in cell migration, which is central to diverse biological phenomena including angiogenesis, wound healing, tumour spread and organ regeneration.