Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.

Inhibition of matrix metalloproteinases blocks lethal hepatitis and apoptosis induced by tumor necrosis factor and allows safe antitumor therapy.

Acute and fulminant liver failure induced by viral hepatitis, alcohol or other hepatotoxic drugs, are associated with tumor necrosis factor (TNF) production. In a mouse model of lethal hepatitis induced by TNF, apoptosis and necrosis of hepatocytes, but also lethality, hypothermia and influx of leukocytes into the liver, are prevented by a broad-spectrum matrix metalloproteinase (MMP) inhibitor, BB-94. Mice deficient in MMP-2, MMP-3 or MMP-9 had lower levels of apoptosis and necrosis of hepatocytes, and better survival. We found induction of MMP-9 activity and fibronectin degradation. Our findings suggest that several MMPs play a critical role in acute, fulminant hepatitis by degrading the extracellular matrix and allowing massive leukocyte influx in the liver. BB-94 also prevented lethality in TNF/interferon-gamma therapy in tumor-bearing mice. A broad-spectrum MMP inhibitor may be potentially useful for the treatment of patients with acute and perhaps chronic liver failure, and in cancer therapies using inflammatory cytokines.

Highly restricted expression of the thymus leukemia antigens on intestinal epithelial cells.

The TL region of the major histocompatibility complex of the mouse contains dozens of tandemly arranged class I genes, including those encoding the thymus leukemia (TL) antigens. TL antigens have been thought to be expressed only on the surface of some T lineage cells, namely immature thymocytes of some mouse strains (TL+ strains), some leukemia cells, and activated T cells. While the function of TL antigens is unknown, recent studies have implicated the products of at least some TL region class I genes as molecules that present antigens to gamma/delta T cells. Since some gamma/delta T cells are known to be specifically associated with certain epithelial tissues, we have investigated the expression of some TL region class I genes in a variety of epithelium-containing tissues. Our results show that the TL antigen gene of C57BL/6 mice, T3b, and the TL antigen genes of BALB/c mice, T3d (previously T3c) and T18d (previously T13c), are highly expressed in the epithelium of the small intestine. In the case of T3b, we further show, using a T3 product-specific antibody, that its product is expressed on the surface of the columnar epithelial cells. In addition, we demonstrated that two other TL region class I genes of C57BL/6 origin, T9b and T21b, are also expressed nearly exclusively in intestinal epithelial cells. These results are consistent with the hypothesis that the products of these TL region class I genes are recognized by gamma/delta T cell receptors of intestinal intraepithelial lymphocytes, a subset of gamma/delta T cells that is localized in the intestinal epithelium and has a restricted V gamma repertoire. Finally, our study indicates that the relative levels of expression of the two homologous TL antigen genes, T3d and T18d, differ widely between the thymus and the intestine.

Resistance to cutaneous graft-vs.-host disease is not induced in T cell receptor delta gene-mutant mice.

The function of murine dendritic epidermal cells (dEC) remains largely speculative, probably because of the lack of a suitable in vivo model, although previous studies suggest that gamma/delta+ dEC may have originally evolved to serve as a self-protection mechanism(s). Our previous study demonstrated that the epidermis of mice that had spontaneously recovered from cutaneous graft-vs-host disease (GVHD) induced by local injection of CD4+ autoreactive T cells contained unexpectedly large numbers of dEC and became resistant to subsequent attempts to induce GVHD in a site-restricted manner, suggesting that the resistance is mediated by dEC. However, because alpha/beta+ dEC as well as gamma/delta+ dEC were greatly increased in number in the epidermis, it was unclear whether gamma/delta+ dEC are indeed responsible for this protection. The availability of this murine model and mice selectively lacking gamma/delta T cells as a result of disruption of the T cell receptor C delta gene segment allowed us to investigate the role of gamma/delta+ dEC. In the epidermis of gamma/delta T cell-deficient mice (delta-/-), a congenital lack of gamma/delta+ dEC was substituted for by alpha/beta+ dEC of either a CD4-8+ or a CD4-8- phenotype. After intradermal injection of the autoreactive T cells, delta-/- mice developed significantly enhanced delayed-type hypersensitivity responses and cutaneous GVHD, which persisted longer than in heterozygous littermate controls (delta+/-). Surprisingly, resistance to the cutaneous GVHD was not induced in the epidermis of delta-/- mice after spontaneous recovery from the GVHD, whereas the "susceptible" epidermis of delta-/+ mice contained large numbers of alpha/beta dEC comparable to those in "resistant" epidermis of delta+/- mice. Injection of day 16 fetal thymocytes from wild-type mice into delta-/- mice resulted in the appearance of donor-type gamma/delta+ dEC in the epidermis, and reconstitution with gamma/delta+ dEC restored the protective immune response of the epidermis against the GVHD to nearly normal levels. These results indicate that gamma/delta+ dEC are responsible for the site-restricted protection against cutaneous GVHD.

Localization of gamma/delta T cells to the intestinal epithelium is independent of normal microbial colonization.

Using monoclonal antibodies identifying all gamma/delta and alpha/beta T cell receptors in cytofluorometric analysis, we have compared the composition of intestinal intraepithelial lymphocytes (i-IEL) in euthymic and athymic germ-free (GF) and conventional (SPF) mice. The results show a marked influence of microbial colonization in the numbers of single-positive (CD4+ or CD8+) alpha/beta i-IEL, but little effect in the pool size or characteristics of gamma/delta i-IEL. In young athymic mice, virtually no alpha/beta i-IEL are detected, while considerable numbers of gamma/delta i-IEL remain, though reduced in GF animals.

Transgenic mice demonstrate that epithelial homing of gamma/delta T cells is determined by cell lineages independent of T cell receptor specificity.

gamma/delta T cells with different TCR repertoires are compartmentalized in different epithelia. This raises the possibility that the TCR-gamma/delta directs homing of T cells to these epithelia. Alternatively, the signals that induce TCR-gamma/delta expression in developing T cells may also induce homing properties in such cells, presumably in the form of cell surface receptors. We have examined this issue by studying the homing of gamma/delta T cells in transgenic mice constructed with specific pairs of rearranged gamma and delta genes. In such mice, most gamma/delta T cells express the transgene-encoded TCR. We find that homing to both skin and gut epithelia is a property of T cells and is not determined by the type of gamma and delta genes used to encode their TCR. We also studied the effect of TCR replacement on the expression of Thy-1 and CD8 proteins on the gamma/delta T cells associated with gut epithelia. Our results show that the expression of the appropriate type of TCR-gamma/delta is not required for the Thy-1 expression by these T cells, suggesting that Thy-1 is not an activation marker. In contrast, CD8 expression by gut gamma/delta T cells seems to depend on the expression of the appropriate type of TCR.

Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis.

Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.

Mice devoid of the glial fibrillary acidic protein develop normally and are susceptible to scrapie prions.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein specifically expressed in astrocytes in the CNS. To examine the function of GFAP in vivo, the Gfap gene was disrupted by gene targeting in embryonic stem cells. Mice homozygous for the mutation were completely devoid of GFAP but exhibited normal development and showed no obvious anatomical abnormalities in the CNS. When inoculated with infectious scrapie prions, the mutant mice exhibited neuropathological changes typical of prion diseases. Infectious prions accumulated in brains of the mutant mice to a degree similar to that in control littermates. These results suggest that GFAP is not essential for the morphogenesis of the CNS or for astrocytic responses against neuronal injury. The results argue against the hypothesis that GFAP plays a crucial role in the pathogenesis of prion diseases.

Facilitation of NMDAR-independent LTP and spatial learning in mutant mice lacking ryanodine receptor type 3.

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.

Deficient cerebellar long-term depression, impaired eyeblink conditioning, and normal motor coordination in GFAP mutant mice.

Mice devoid of glial fibrillary acidic protein (GFAP), an intermediate filament protein specifically expressed in astrocytes, develop normally and do not show any detectable abnormalities in the anatomy of the brain. In the cerebellum, excitatory synaptic transmission from parallel fibers (PFs) or climbing fibers (CFs) to Purkinje cells is unaltered, and these synapses display normal short-term synaptic plasticity to paired stimuli in GFAP mutant mice. In contrast, long-term depression (LTD) at PF-Purkinje cell synapses is clearly deficient. Furthermore, GFAP mutant mice exhibited a significant impairment of eyeblink conditioning without any detectable deficits in motor coordination tasks. These results suggest that GFAP is required for communications between Bergmann glia and Purkinje cells during LTD induction and maintenance. The data support the notion that cerebellar LTD is a cellular mechanism closely associated with eyeblink conditioning, but is not essential for motor coordination tasks tested.

Cortical glutamatergic neurons mediate the motor sedative action of diazepam.

The neuronal circuits mediating the sedative action of diazepam are unknown. Although the motor-depressant action of diazepam is suppressed in alpha1(H101R) homozygous knockin mice expressing diazepam-insensitive alpha1-GABA(A) receptors, global alpha1-knockout mice show greater motor sedation with diazepam. To clarify this paradox, attributed to compensatory up-regulation of the alpha2 and alpha3 subunits, and to further identify the neuronal circuits supporting diazepam-induced sedation, we generated Emx1-cre-recombinase-mediated conditional mutant mice, selectively lacking the alpha1 subunit (forebrain-specific alpha1(-/-)) or expressing either a single wild-type (H) or a single point-mutated (R) alpha1 allele (forebrain-specific alpha1(-/H) and alpha1(-/R) mice, respectively) in forebrain glutamatergic neurons. In the rest of the brain, alpha1(-/R) mutants are heterozygous alpha1(H101R) mice. Forebrain-specific alpha1(-/-) mice showed enhanced diazepam-induced motor depression and increased expression of the alpha2 and alpha3 subunits in the neocortex and hippocampus, in comparison with their pseudo-wild-type littermates. Forebrain-specific alpha1(-/R) mice were less sensitive than alpha1(-/H) mice to the motor-depressing action of diazepam, but each of these conditional mutants had a similar behavioral response as their corresponding control littermates. Unexpectedly, expression of the alpha1 subunit was reduced in forebrain, notably in alpha1(-/R) mice, and the alpha3 subunit was up-regulated in neocortex, indicating that proper alpha1 subunit expression requires both alleles. In conclusion, conditional manipulation of GABA(A) receptor alpha1 subunit expression can induce compensatory changes in the affected areas. Specifically, alterations in GABA(A) receptor expression restricted to forebrain glutamatergic neurons reproduce the behavioral effects seen after a global alteration, thereby implicating these neurons in the motor-sedative effect of diazepam.

JDP2 suppresses adipocyte differentiation by regulating histone acetylation.

Among the events that control cellular differentiation, the acetylation of histones plays a critical role in the regulation of transcription and the modification of chromatin. Jun dimerization protein 2 (JDP2), a member of the AP-1 family, is an inhibitor of such acetylation and contributes to the maintenance of chromatin structure. In an examination of Jdp2 'knock-out' (KO) mice, we observed elevated numbers of white adipocytes and significant accumulation of lipid in the adipose tissue in sections of scapulae. In addition, mouse embryo fibroblasts (MEFs) from Jdp2 KO mice were more susceptible to adipocyte differentiation in response to hormonal induction and members of the CCAAT/enhancer-binding proteins (C/EBP) gene family were expressed at levels higher than MEFs from wild-type mice. Furthermore, JDP2 inhibited both the acetylation of histone H3 in the promoter of the gene for C/EBPdelta and transcription from this promoter. Our data indicate that JDP2 plays a key role as a repressor of adipocyte differentiation by regulating the expression of the gene for C/EBPdelta via inhibition of histone acetylation.

Schizophrenia-like phenotypes in mice with NMDA receptor ablation in intralaminar thalamic nucleus cells and gene therapy-based reversal in adults.

In understanding the mechanism of schizophrenia pathogenesis, a significant finding is that drug abuse of phencyclidine or its analog ketamine causes symptoms similar to schizophrenia. Such drug effects are triggered even by administration at post-adolescent stages. Both drugs are N-methyl-d-aspartate receptor (NMDAR) antagonists, leading to a major hypothesis that glutamate hypofunction underlies schizophrenia pathogenesis. The precise region that depends on NMDAR function, however, is unclear. Here, we developed a mouse strain in which NMDARs in the intralaminar thalamic nuclei (ILN) were selectively disrupted. The mutant mice exhibited various schizophrenia-like phenotypes, including deficits in working memory, long-term spatial memory, and attention, as well as impulsivity, impaired prepulse inhibition, hyperlocomotion and hyperarousal. The electroencephalography analysis revealed that the mutant mice had a significantly reduced power in a wide range of frequencies including the alpha, beta and gamma bands, both during wake and rapid eye movement (REM) sleep, and a modest decrease of gamma power during non-REM sleep. Notably, restoring NMDARs in the adult ILN rescued some of the behavioral abnormalities. These findings suggest that NMDAR dysfunction in the ILN contributes to the pathophysiology of schizophrenia-related disorders. Furthermore, the reversal of inherent schizophrenia-like phenotypes in the adult mutant mice supports that ILN is a potential target site for a therapeutic strategy.

Memory trace of motor learning shifts transsynaptically from cerebellar cortex to nuclei for consolidation.

Adaptation of ocular reflexes is a prototype of motor learning. While the cerebellum is acknowledged as the critical site for motor learning, the functional differences between the cerebellar cortex and nuclei in motor memory formation are not precisely known. Two different views are proposed: one that the memory is formed within the cerebellar flocculus, and the other that the memory is formed within vestibular nuclei. Here we developed a new paradigm of long-term adaptation of mouse horizontal optokinetic response eye movements and examined the location of its memory trace. We also tested the role of flocculus and inferior olive in long-term adaptation by chronic lesion experiments. Reversible bilateral flocculus shutdown with local application of 0.5 microl-5% lidocaine extinguished the memory trace of day-long adaptation, while it very little affected the memory trace of week-long adaptation. The responsiveness of vestibular nuclei after week-long adaptation was examined by measuring the extracellular field responses to the electrical stimulation of vestibular nerve under trichloroacetaldehyde anesthesia. The amplitudes and slopes of evoked monosynaptic field response (N1) of week-long adapted mice were enhanced around the medial vestibular nucleus compared with those of control mice. Chronic flocculus or inferior olive lesions abolished both day and week-long adaptations. These results suggest that the functional memory trace of short-term adaptation is formed initially within the cerebellar cortex, and later transferred to vestibular nuclei to be consolidated to a long-term memory. Both day and week-long adaptations were markedly depressed when neural nitric oxide was pharmacologically blocked locally and when neuronal nitric oxide synthase was ablated by gene knockout, suggesting that cerebellar long-term depression underlies both acquisition and consolidation of motor memory.

Reduced angiogenesis and tumor progression in gelatinase A-deficient mice.

Matrix proteolysis is thought to play a crucial role in several stages of tumor progression, including angiogenesis, and the invasion and metastasis of tumor cells. We investigated the specific role of gelatinase A (matrix metalloproteinase 2) on these events using gelatinase A-deficient mice. In these mice, tumor-induced angiogenesis was suppressed according to dorsal air sac assay. When B16-BL6 melanoma cells or Lewis lung carcinoma cells were implanted intradermally, the tumor volumes at 3 weeks after implantation in the gelatinase A-deficient mice decreased by 39% for B16-BL6 melanoma and by 24% for Lewis lung carcinoma (P < 0.03 for each tumor). The number of lung colonies of i.v. injections fell by 54% for B16-BL6 melanoma and 77% for Lewis lung carcinoma (P < 0.014 and P < 0.0015, respectively). These results indicated that host-derived gelatinase A plays an important role in angiogenesis and tumor progression, suggesting the usefulness of gelatinase A inhibitors for anticancer chemotherapy.

Learning induces a CDC2-related protein kinase, KKIAMRE.

To elucidate molecular mechanisms in learning and memory, we analyzed expression of mRNAs in brains of rabbits undergoing eyeblink conditioning. Infusion of the transcription inhibitor actinomycin D into the cerebellar interpositus nucleus reversibly blocked learning but not performance of the conditioned response. Differential display PCR analysis of cerebellar interpositus RNAs from trained and pseudotrained rabbits identified a 207 bp band that was induced with learning. The fragment was used to isolate a cDNA from a lambdagt11 rabbit brain library containing a 1698 bp open reading frame. The deduced amino acid sequence contains the KKIAMRE motif, which is conserved among cell division cycle 2 (cdc2)-related kinases. These results suggest that there is a new category of cdc2-related kinases in the brain whose function may be important in learning and memory.

Strong expression of NETRIN-G2 in the monkey claustrum.

The claustrum is a phylogenetically conserved structure, with extensive reciprocal connections with cortical regions, and has thus been considered important for sensory, motor, emotional, and mnemonic coordination or integration. Here, we show by in situ hybridization that the adult monkey claustrum is strongly positive for NETRIN-G2, a gene encoding a glycosyl phosphatidyl-inositol-linked membrane protein, which constitutes a subfamily with NETRIN-G1 within the netrin/UNC6 family. There is a conspicuous dorsal/ventral differentiation, where the label is stronger in the ventral claustrum. NETRIN-G2 positive neurons are not GABAergic, but rather correspond to claustrocortical projection neurons, as demonstrated by retrograde transport of Fast Blue from cortical injections and by double in situ hybridization for NETRIN-G2 and GAD67. Since NETRIN-G2 is known to be preferentially expressed in cortex, in contrast with the thalamically expressed NETRIN-G1, these results raise the possibility of some functional similarity in regulation of excitatory neural transmission in the claustrum and cortex.

Thalamic adenylyl cyclase 1 is required for barrel formation in the somatosensory cortex.

Cyclic AMP signaling is critical for activity-dependent refinement of neuronal circuits. Global disruption of adenylyl cyclase 1 (AC1), the major calcium/calmodulin-stimulated adenylyl cyclase in the brain, impairs formation of whisker-related discrete neural modules (the barrels) in cortical layer 4 in mice. Since AC1 is expressed both in the thalamus and the neocortex, the question of whether pre- or postsynaptic (or both) AC1 plays a role in barrel formation has emerged. Previously, we generated cortex-specific AC1 knockout (Cx-AC1KO) mice and found that these animals develop histologically normal barrels, suggesting a potentially more prominent role for thalamic AC1 in barrel formation. To determine this, we generated three new lines of mice: one in which AC1 is disrupted in nearly half of the thalamic ventrobasal nucleus cells in addition to the cortical excitatory neurons (Cx/pTh-AC1KO mouse), and another in which AC1 is disrupted in the thalamus but not in the cortex or brainstem nuclei of the somatosensory system (Th-AC1KO mouse). Cx/pTh-AC1KO mice show severe deficits in barrel formation. Th-AC1KO mice show even more severe disruption in barrel patterning. In these two lines, single thalamocortical (TC) axon labeling revealed a larger lateral extent of TC axons in layer 4 compared to controls. In the third line, all calcium-stimulated adenylyl cyclases (both AC1 and AC8) are deleted in cortical excitatory neurons. These mice have normal barrels. Taken together, these results indicate that thalamic AC1 plays a major role in patterning and refinement of the mouse TC circuitry.

Diminished corneal angiogenesis in gelatinase A-deficient mice.

The goal of the present study was to define the role of gelatinase A in angiogenesis. We performed corneal micropocket assays in gelatinase A-deficient mice and their age-matched wild-type littermates. The corneal neovascular area in gelatinase A-deficient mice (0.15+/-0.14 mm(2)) was significantly less than that of wild-type littermates (0.53+/-0.35 mm(2); P<0.01). Similarly, aortic ring assays showed significant reduction of endothelial outgrowth in gelatinase A-deficient mice (0.26+/-0.14 mm(2)) as compared to wild-type littermates (0.44+/-0.06 mm(2); P<0.05). These results suggest that gelatinase A may play an important role in the regulation of corneal angiogenesis.

Difficulty in detecting in vivo extracellular infective virus in cattle naturally infected with bovine leukemia virus.

A search was made for the presence in vivo of infective bovine leukemia virus (BLV) in secretions and excretions as well as in several tissues from five bulls, three cows and one calf. Two of the bulls and three of the cows were natural tumour cases of enzootic bovine leukosis, another three of the bulls were natural cases of persistent lymphocytosis and the calf was a natural tumour case of juvenile bovine leukosis. BLV infection was confirmed for all cattle, except the juvenile leukosis case, by detection of BLV-specific agar gel immunodiffusion (AGID) antibodies. Cell-free preparations were made from homogenates of lymphocytes, lymph nodes, spleens, livers, intestines, urinary bladders, salivary glands, mammary glands, pools of prostate glands and testicles and feces as well as from plasma, urine and milk, by passing them through 5 micrometers membrane filters. BLV infectivity in these cell-free preparations was examined by syncytia infectivity assay using susceptible cell cultures of bovine or ovine origin. Infective BLV could not be isolated from any of these cell-free preparations of plasma, secretions, excretions and tissues, although it was isolated consistently from the in vitro cultures of viable lymphocytes obtained from BLV-infected cattle. There was no indication of BLV involvement in the case of juvenile bovine leukosis.