Hyperphagia and leptin resistance in tissue inhibitor of metalloproteinase-2 deficient mice.

Obesity is a complex genetic and behavioural disorder arising from the improper integration of peripheral signals at central autonomic centres. For the hypothalamus to respond to dynamic physiological alterations, it must retain a degree of plasticity throughout life. Evidence is mounting that an intricate balance between matrix metalloproteinase (MMP)-mediated extracellular matrix proteolysis and tissue inhibitor of metalloproteinase (TIMP)-mediated proteolysis inhibition contributes to tissue remodelling. However, few studies have examined the role of MMPs/TIMPs in hypothalamic remodelling and energy homeostasis. To determine the contribution of TIMP-2 to the hypothalamic regulation of feeding, body mass and food consumption were monitored in TIMP-2 knockout (KO) mice fed a standard chow or high-fat diet (HFD). TIMP-2 KO mice of both sexes gained more weight than wild-type (WT) mice, even when fed the chow diet. Before the onset of obesity, TIMP-2 KO mice were hyperphagic, without increased orexigenic or decreased anorexigenic neuropeptide expression, but leptin resistant (i.e. reduced leptin-induced anorexigenic response and signal transducer and activator of transcription 3 activation). HFD exacerbated weight gain and hyperleptinaemia. In addition, proteolysis was increased in the arcuate nucleus of TIMP-2 KO mice. These data suggest a role for TIMP-2 in hypothalamic control of feeding and energy homeostasis.

Differential spatial distribution and temporal regulation of tissue inhibitor of metalloproteinase mRNA expression during rat central nervous system development.

The elucidation of the cellular and molecular mechanisms governing the maturation of the central nervous system (CNS) is rapidly emerging. Cell-cell and cell-matrix interactions play critical roles in all phases of developmental tissue remodeling. Throughout development, an intricate balance between extracellular matrix synthesis and degradation is preserved by the opposing actions of matrix metalloproteinases (MMPs) and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Although recent evidence suggests that TIMPs exert diverse cell biological functions distinct from their MMP-inhibitory activities, few studies have investigated MMP or TIMP expression during CNS development. The present report analyzes the mRNA expression of the four known TIMPs throughout the course of embryonic and postnatal rat CNS development. The results clearly demonstrate the unique spatial distribution and temporal regulation of TIMP expression and suggest a distinct role for each TIMP during CNS development.

Regulation of tissue inhibitor of metalloproteinase-3 (Timp-3) mRNA expression during rat CNS development.

To preserve tissue integrity during the structural rearrangements that occur during central nervous system (CNS) development, an intricate balance between extracellular matrix (ECM) synthesis and degradation must be maintained. The matrix metalloproteinases (MMPs) are believed to be the main mediators of ECM degradation. Because MMPs function in the turnover of a broad-spectrum of ECM proteins their activity is tightly regulated by interaction with tissue inhibitors of metalloproteinases (TIMPs). Whereas the primary function of TIMPs is to inhibit MMP activity, evidence is mounting that TIMPs are multifunctional molecules that exert diverse cell biological functions distinct from their MMP-inhibitory activities. Although the role of MMPs and TIMPs in the morphogenesis of non-neural tissues has been investigated, to date few studies have analyzed MMP or TIMP expression during CNS development. In the present report, we demonstrate the regulation of Timp-3 mRNA expression throughout the course of CNS development. In particular, Timp-3 mRNA is expressed in embryonic ventricular zones and the postnatal subventricular zone (SVZ). In addition, Timp-3 is expressed in the rostral migratory steam (RMS) to the olfactory bulb in a pattern similar to the ECM proteoglycan brevican. These data suggest that TIMP-3 and brevican may act in concert to guide neuronal migration along the RMS.

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP-selective receptor in cultured rat astrocytes, human brain tumors, and in response to acute intracranial injury.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse activities in the nervous system. In addition to its more classic role as a neurotransmitter, PACAP functions as a neurotrophic factor. PACAP exerts these activities by binding to PACAP-selective (PAC1) or nonselective (VPAC1, VPAC2) receptors (-R). Glial cells also exhibit PACAP binding, which is associated with the increased proliferation of astrocytes. The present report demonstrates a distinct spatiotemporal regulation of PACAP, PAC1-R, VPAC1-R, and VPAC2-R expression in primary cultured rat astrocytes. To determine the role of PACAP and PAC1-R expression on glial proliferation, two in vivo models were examined--human brain tumors of glial origin and the reactive gliosis induced by a penetrating stab wound to the mature rat brain. Relative to normal human brain, PAC1-R expression is significantly upregulated in glioma, particularly oligodendrogliomas. While similar polymerase chain reaction (PCR) analysis does not detect PACAP expression, in situ hybridization studies reveal PACAP expression in a limited number of cells within the tumor. In sharp contrast, neither PACAP nor PAC1-R expression are upregulated consequent to injury. These results suggest a distinct role for PACAP and PAC1-R in glioma development and nervous system response to injury.

Developmental regulation of membrane type-5 matrix metalloproteinase (MT5-MMP) expression in the rat nervous system.

An intricate balance between extracellular matrix (ECM) synthesis and degradation must be maintained during developmental tissue remodeling. Matrix metalloproteinases (MMPs) are the main mediators of ECM degradation. A subset of MMPs, referred to as membrane-type MMPs, contains a transmembrane domain that restricts protease activity at the cell surface. Membrane type-5 MMP is predominantly expressed in the brain. The present report is the first to demonstrate the temporal regulation and spatial distribution of MT5-MMP mRNA during nervous system development.

Developmental regulation of pituitary adenylate cyclase-activating polypeptide and PAC(1) receptor mRNA expression in the rat central nervous system.

As the brain develops, a homogeneous population of mitotically active progenitors generates the molecularly heterogeneous post-mitotic cells of the mature brain. The balance between cell division, growth arrest and differentiation of these progenitors undoubtedly requires the activation of a vast array of genes. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. Within the nervous system, PACAP has been shown to stimulate neurite outgrowth, regulate neurotransmitter production and neuronal survival. These diverse biological actions are mediated through interaction with two types of receptors, a PACAP-selective receptor (PAC(1)-R) and receptors which interact almost equally with both VIP and PACAP. Since several lines of evidence suggest that PACAP acts as a neurotrophic factor, we sought to characterize PACAP and PAC(1)-R expression in the developing rat nervous system. The PAC(1)-R is expressed at very high levels in ventricular zones throughout the neuraxis. In addition to the embryonic enrichment in proliferative zones, PAC(1)-R expression is maintained in areas of neurogenesis in the adult central nervous system (CNS), namely, the subventricular zone of the olfactory bulb and hippocampal dentate gyrus. In contrast, PACAP is expressed primarily in the post-mitotic parenchyma. This temporal regulation and cellular distribution suggests that PACAP, through its interaction with the PAC(1)-R, may play a role in mammalian neurogenesis.

Differential regulation of tissue inhibitor of metalloproteinase mRNA expression in response to intracranial injury.

Injury to the CNS induces complex cellular and molecular interactions referred to as reactive gliosis. Alterations in the extracellular microenvironment associated with the gliotic response are believed to be the primary cause of regenerative failure of the mature CNS. For injured neurons to reestablish severed connections their processes must explore the extracellular milieu. Thus far, experiments have focused on extracellular matrix (ECM) proteins whose expression is upregulated after CNS injury and that exert inhibitory effects on neurite outgrowth. An intricate balance between ECM synthesis and degradation must be maintained during the tissue remodeling associated with injury. Matrix metalloproteinases (MMPs) are believed to be the main mediators of ECM degradation. MMP activity is tightly regulated by interaction with tissue inhibitors of metalloproteinases (TIMPs). To determine whether TIMPs are expressed during injury-induced matrix remodeling, TIMP expression was examined during reactive gliosis. A stab injury to the mature rat brain leads to the differential regulation of TIMP mRNA expression. Timp-1 and Timp-2 mRNA are significantly upregulated after injury, while the expression of Timp-3 and Timp-4 is unaltered. The expression of Timp-1 in reactive astrocytes and Timp-2 in microglia and neurons suggests these TIMPs may serve distinct functions in response to injury.

Impairment of the blood-brain barrier: a potential surrogate delineating the determinants of cerebral bleeding caused by fibrinolytic drugs.

BACKGROUND: Intracranial bleeding is encountered in some patients with acute myocardial infarction treated with fibrinolytic drugs, and especially in patients with occult cerebral vasculopathy. In order to determine whether pharmacologically induced plasminemia is a determinant, and whether impairment of the blood-brain barrier can serve as a marker of risk, we studied spontaneously hypertensive stroke-prone rats (SHRSP) genetically disposed to cerebral vasculopathy. METHODS: In order to simulate the induction of plasminemia in patients treated with fibrinolytic drugs for acute myocardial infarction, three intravenous injections of human plasminogen and human tissue-type plasminogen activator (t-PA) were administered to the rats at 2-h intervals (12 mg plasminogen plus 60 micrograms t-PA, 6 mg plus 30 micrograms t-PA, and 0.5 mg plus 2.5 micrograms t-PA), and serial blood samples were assayed for plasmin. One hour after the final intravenous injection, the brain was perfusion-fixed with 4% formaldehyde, and the blood-brain barrier integrity was assessed by localization of immunoglobulin G (IgG) using fluorescein-conjugated goat anti-rat IgG and confocal microscopy. RESULTS: Transient plasminemia followed each injection of plasminogen and t-PA. Intracranial extravasation of IgG was observed in nine of 11 SHRSP treated with t-PA and plasminogen. None of seven SHRSP injected with vehicle alone exhibited extravasation (chi 2 = 6.37, P = 0.01). CONCLUSIONS: The results indicate that the blood-brain barrier in SHRSP is susceptible to impairment secondary to pharmacologically induced plasminemia and is predisposed to bleeding. The preparation developed may facilitate the delineation of relative risk of injury to cerebral vessels with thrombolytic therapy.

Intracranial injury acutely induces the expression of the secreted isoform of the CNS-specific hyaluronan-binding protein BEHAB/brevican.

Hyaluronan (HA) plays an important role in tissue reorganization in response to injury. The mechanisms by which HA participates in these processes are likely to include HA-binding proteins. Previously, we reported the cloning and initial characterization of a central nervous system (CNS)-specific HA-binding protein, BEHAB (brain enriched hyaluronan binding), which was independently cloned in another laboratory and named brevican. BEHAB/brevican mRNA is expressed in the ventricular zone coincident with the initial proliferation and migration of glial cells and in surgical samples of human glioma, where glial-derived cells proliferate and migrate. To determine whether BEHAB/brevican is also expressed during the cellular proliferation and migration associated with CNS injury, we have examined BEHAB/brevican expression during reactive gliosis. BEHAB/brevican occurs as secreted and cell-surface, glycosylphosphatidylinositol (GPI)-anchored, isoforms. The secreted, but not the GPI-anchored, isoform is up-regulated in response to a stab wound to the adult rat brain. The temporal regulation and spatial distribution of BEHAB/brevican expression parallel the gliotic response and the expression of the intermediate filament protein nestin. The up-regulation of BEHAB/brevican in response to CNS injury suggests a role for this extracellular matrix molecule in reactive gliosis. Glial process extension, a central element in the glial response to injury, may require the reexpression of both cytoskeletal and matrix elements that are normally expressed during the glial motility seen in the immature brain.

Expression of a cleaved brain-specific extracellular matrix protein mediates glioma cell invasion In vivo.

Malignant gliomas (primary brain tumors) aggressively invade the surrounding normal brain. This invasive ability is not demonstrated by brain metastases of nonglial cancers. The brain-specific, brain-enriched hyaluronan binding (BEHAB)/brevican gene, which encodes an extracellular hyaluronan-binding protein, is consistently expressed by human glioma and is not expressed by tumors of nonglial origin (Jaworski et al., 1996). BEHAB/brevican can be cleaved into an N-terminal fragment that contains a hyaluronan-binding domain (HABD) and a C-terminal fragment (Yamada et al., 1995). Here, using antisera to peptides in the predicted N-terminal and C-terminal proteolytic fragments, we demonstrate that the BEHAB/brevican protein is cleaved in invasive human and rodent gliomas. A role for this protein in glioma cell invasion was tested by transfecting a noninvasive cell line with the BEHAB/brevican gene. The noninvasive 9L glioma cell was transfected with either full-length BEHAB/brevican or the HABD and tested for invasion in in vitro and in vivo invasion assays. Although both constructs increased invasion in vitro, only the HABD increased invasion by tumors growing in vivo. Experimental intracranial tumors from full-length transfectants showed no increase in invasion over control tumors, whereas tumors from HABD transfectants showed a marked potentiation of tumor invasion, producing new tumor foci at sites distant from the main tumor mass. This work demonstrates a role for a brain-specific extracellular matrix protein in glioma invasion, opening new therapeutic avenues for a uniformly fatal disease.

BEHAB (brain enriched hyaluronan binding) is expressed in surgical samples of glioma and in intracranial grafts of invasive glioma cell lines.

Malignant gliomas aggressively invade the surrounding normal brain, whereas brain metastases of nonglial tumors do not. The invasive behavior of gliomas may be mediated by tissue- or tumor-specific extracellular proteins. mRNA for the brain-specific extracellular brain enriched hyaluronan-binding protein (BEHAB) is not detectable in normal adult human cortex or in any nonglioma tumor examined. BEHAB is consistently expressed in surgical samples of glioma (n = 27). Glioma cell lines maintained under standard cell culture conditions or grown as s.c. tumors do not express BEHAB. When grown as intracranial grafts, glioma cell lines that invade the brain express BEHAB, whereas noninvasive cell lines do not. BEHAB is a unique and selective marker for glioma and may play a role in tumor invasion.

The CNS-specific hyaluronan-binding protein BEHAB is expressed in ventricular zones coincident with gliogenesis.

Hyaluronan (HA) is a ubiquitous component of extracellular matrices, and in several systems it plays a central role in regulating cellular proliferation and differentiation. Cell, or tissue,-specific functions of HA are likely to be mediated by cell, or tissue,-specific HA-binding proteins. We previously hyaluronan-binding protein from rat and cat (Jaworski et al., 1994). In view of the potential role of HA in neural differentiation, we examined the expression of BEHAB during late embryonic and early postnatal development of the rat. BEHAB is expressed at very high levels in ventricular zones throughout the neuraxis. Expression is first detected at embryonic day 15 (E15) in the spinal cord, and is detected at progressively more rostral levels at later ages. BEHAB expression, like other features of neural development, follows both caudal-to-rostral and ventral-to-dorsal gradients. The timing of BEHAB expression parallels the timing of the generation of glial cells. In all areas of the CNS examined, BEHAB expression begins after the peak of neurogenesis and coincident with gliogenesis. The regulation of proliferation and differentiation by HA in other tissues, together with the expression of BEHAB in zones of mitotic activity coincident with the generation of glia, suggests that the extracellular matrix protein encoded by BEHAB could play a role in the generation or differentiation of CNS glia.

BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain.

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix of all tissues. In the mammalian central nervous system (CNS) HA is present throughout development and into adulthood. While the functions of HA are likely to be mediated by HA-binding proteins, no cell or tissue specific HA-binding proteins have been reported. In an effort to characterize the composition of the extracellular matrix of the CNS, we sought to identify neural HA-binding proteins. We report here the isolation and characterization of a cDNA with a high degree of sequence homology to members of the proteoglycan tandem repeat (PTR) family of HA-binding proteins. Unlike other HA-binding proteins, the expression of this cDNA is restricted to the CNS. We propose the name BEHAB, Brain Enriched HyAluronan Binding protein, for this gene. The expression of BEHAB mRNA is developmentally regulated; expression is first detected in the late embryonic period and peaks during the first two postnatal weeks. In the embryo, BEHAB is expressed at highest levels in mitotically active cells. The sequence of BEHAB has long stretches of identity between rat and cat, suggesting that the encoded protein is functionally important. The size and sequence of BEHAB are consistent with the possibility that it could serve a function like link protein, stabilizing interactions between HA and brain proteoglycans. These observations suggest that existence of other tissue specific HA-binding proteins.