SPARC/Osteonectin mRNA Is Induced in Blood Vessels Following Injury to the Adult Rat Cerebral Cortex

Recently we described the pattern of expression of the anti-adhesive glycoprotein SPARC/osteonectin in the developing and adult brain. SPARC mRNA was present in developing blood vessels during neurogenesis, but was not detected in the mature vasculature. We have now examined the effect of a lesion to the adult rat cerebral cortex on the expression of SPARC by in situ hybridization. SPARC mRNA was increased in the zone proximal to the wound at 3 to 10 days after cortical brain injury. During this period, SPARC was induced in mature blood vessels close to the lesion site and in blood vessels which develop following injury. These results suggest a role for SPARC in the process of angiogenesis following injury to the adult cerebral cortex.     

Induction of a heat shock gene at the site of tissue injury in the rat brain

Our objective was to investigate whether localized tissue injury induces expression of a gene encoding the major 70 kd heat shock protein (hsp70) in the mammalian nervous system. A small surgical cut was made in the rat cerebral cortex. By 2 hr postsurgery a dramatic and highly localized induction of hsp70 mRNA was detected at the lesion site using in situ hybridization with labeled riboprobe. By 12 hr the intensity of the signal had diminished, and by 24 hr only a few cells along the walls of the cut demonstrated a high level of hsp70 mRNA. Both neurons and glial cells at the site of the surgical cut responded to tissue injury by induction of hsp70 mRNA. Induction was not observed in other brain regions, nor was the pattern of constitutive expression affected by the surgical procedure.     

The role of SPARC in extracellular matrix assembly

SPARC is a collagen-binding matricellular protein. Expression of SPARC in adult tissues is frequently associated with excessive deposition of collagen and SPARC-null mice fail to generate a robust fibrotic response to a variety of stimuli. This review summarizes recent advancements in the characterization of the binding of SPARC to collagens and describes the results of studies that implicate a function for SPARC in the regulation of the assembly of basal lamina and fibrillar collagen in the ECM. Potential cellular mechanisms that underlie SPARC activity in ECM deposition are also explored.     

Rapid, widespread, and longlasting induction of nestin contributes to the generation of glial scar tissue after CNS injury

Neuronal regeneration does generally not occur in the central nervous system (CNS) after injury, which has been attributed to the generation of glial scar tissue. In this report we show that the composition of the glial scar after traumatic CNS injury in rat and mouse is more complex than previously assumed: expression of the intermediate filament nestin is induced in reactive astrocytes. Nestin induction occurs within 48 hours in the spinal cord both at the site of lesion and in degenerating tracts and lasts for at least 13 months. Nestin expression is induced with similar kinetics in the crushed optic nerve. In addition to the expression in reactive astrocytes, we also observed nestin induction within 48 hours after injury in cells close to the central canal in the spinal cord, while nestin expressing cells at later timepoints were found progressively further out from the central canal. This dynamic pattern of nestin induction after injury was mimicked by lacZ expressing cells in nestin promoter/lacZ transgenic mice, suggesting that defined nestin regulatory regions mediate the injury response. We discuss the possibility that the spatiotemporal pattern of nestin expression reflects a population of nestin positive cells, which proliferates and migrates from a region close to the central canal to the site of lesion in response to injury.     

Enhanced growth of pancreatic tumors in SPARC-null mice is associated with decreased deposition of extracellular matrix and reduced tumor cell apoptosis

SPARC, a matricellular glycoprotein, modulates cellular interaction with the extracellular matrix (ECM). Tumor growth and metastasis occur in the context of the ECM, the levels and deposition of which are controlled in part by SPARC. Tumor-derived SPARC is reported to stimulate or retard tumor progression depending on the tumor type, whereas the function of host-derived SPARC in tumorigenesis has not been explored fully. To evaluate the function of endogenous SPARC, we have examined the growth of pancreatic tumors in SPARC-null (SP(-/-)) mice and their wild-type (SP(+/+)) counterparts. Mouse pancreatic adenocarcinoma cells injected s.c. grew significantly faster in SP(-/-) mice than cells injected into SP(+/+) animals, with mean tumor weights at sacrifice of 0.415 +/- 0.08 and 0.086 +/- 0.03 g (P < 0.01), respectively. Lack of endogenous SPARC resulted in decreased collagen deposition and fiber formation, alterations in the distribution of tumor-infiltrating macrophages, and decreased tumor cell apoptosis. There was no difference in microvessel density of tumors from SP(-/-) or SP(+/+) mice. However, tumors grown in SP(-/-) had a lower percentage of blood vessels that expressed smooth muscle alpha-actin, a marker of pericytes. These data reflect the importance of ECM deposition in regulating tumor growth and demonstrate that host-derived SPARC is a critical factor in the response of host tissue to tumorigenesis.     

SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway

SPARC (osteonectin/BM40) is a secreted protein that modifies the interaction of cells with extracellular matrix (ECM). When we added SPARC to cultured rabbit synovial fibroblasts and analyzed the secreted proteins, we observed an increase in the expression of three metalloproteinases--collagenase, stromelysin, and the 92-kD gelatinase-- that together can degrade both interstitial and basement membrane matrices. We further characterized the regulation of one of these metalloproteinases, collagenase, and showed that both collagenase mRNA and protein are upregulated in fibroblasts treated with SPARC. Experiments with synthetic SPARC peptides indicated that a region in the neutral alpha-helical domain III of the SPARC molecule, which previously had no described function, was involved in the regulation of collagenase expression by SPARC. A sequence in the carboxyl-terminal Ca(2+)-binding domain IV exhibited similar activity, but to a lesser extent. SPARC induced collagenase expression in cells plated on collagen types I, II, III, and V, and vitronectin, but not on collagen type IV. SPARC also increased collagenase expression in fibroblasts plated on ECM produced by smooth muscle cells, but not in fibroblasts plated on a basement membrane-like ECM from Engelbreth-Holm-Swarm sarcoma. Collagenase was induced within 4 h in cells treated with phorbol diesters or plated on fibronectin fragments, but was induced after 8 h in cells treated with SPARC. A number of proteins were transiently secreted by SPARC-treated cells within 6 h of treatment. Conditioned medium that was harvested from cultures 7 h after the addition of SPARC, and depleted of residual SPARC, induced collagenase expression in untreated fibroblasts; thus, part of the regulation of collagenase expression by SPARC appears to be indirect and proceeds through a secreted intermediate. Because the interactions of cells with ECM play an important role in regulation of cell behavior and tissue morphogenesis, these results suggest that molecules like SPARC are important in modulating tissue remodeling and cell-ECM interactions.     

The studies on neurogenesis induced by brain injury in adult ring dove

INTRODUCTIONItisacommonstatementthatillhighervertebratestheCNScompletesneuronaldevelopmentduringthepre--andprenatalperiods.Preliminaryworkshowedthatneurogenesisconfinuedinthebrainofsongbirdduringadulthood.Manynewbornneuronsareincorporatedintothevocal-cont…     

Changes in expression of neuronal and glial glutamate transporters in lead-exposed adult rat brain

Excitatory amino acid transporters (EAATs) are membrane-bound proteins localized in glial and neuronal cells which transport glutamate (Glu) in a process essential for terminating its action and protecting neurons from excitotoxic damage. Since Pb-induced neurotoxicity has a glutamatergic component and astrocytes serve as a cellular Pb deposition site, it was of interest to investigate the response of main glutamate transporters to short-term lead exposure in the adult rat brain (25mg/kg b.w. of lead acetate, i.p. for 3 days). We examined the expression of mRNA and protein of GLAST, GLT-1 and EAAC1 in homogenates obtained from cerebellum, hippocampus and forebrain. Molecular evidence is provided which indicates that, of the two glial transporters, GLT-1 is more susceptible than GLAST to the neurotoxic effect arising from Pb. RT-PCR analysis revealed highly decreased expression of GLT-1 mRNA in forebrain and hippocampus. In contrast, GLAST was overexpressed in forebrain and in cerebellum. In the case of EAAC1, the enhanced expression of mRNA and protein of transporter was observed only in forebrain. The results demonstrate regional differences in the expression of glutamate transporters after short-term exposure to Pb. In forebrain, downregulation of GLT-1 is compensated by enhanced expression of GLAST, while in hippocampus, the expression of both is lowered. This observation suggests that under conditions of Pb toxicity in adult rat brain, the hippocampus is most vulnerable to the excitotoxic cell damage arising from impaired clearance of the released glutamate.     

Molecular cloning of SC1: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/BM40/SPARC.

We describe the cloning of SC1, a novel cDNA that was selected from a rat brain expression library using a mixed polyclonal antibody directed against synaptic junction glycoproteins. SC1 detects a 3.2 kb mRNA expressed throughout postnatal development of the brain and present at high levels in the adult. In situ hybridization reveals that the SC1 mRNA is expressed widely in the brain and is present in many types of neurons. DNA sequence data suggest that the SC1 product is a secreted, calcium binding glycoprotein. Strikingly, the carboxy-terminal region of the SC1 protein shows substantial similarity to the extracellular matrix glycoprotein osteonectin/BM40/SPARC. These data are consistent with the hypothesis that SC1 is an extracellular matrix glycoprotein in the brain.     

SPARC downregulation attenuates the profibrogenic response of hepatic stellate cells induced by TGF-β1 and PDGF

Liver fibrosis is an active process that involves changes in cell-cell and cell-extracellular matrix (ECM) interaction. Secreted protein, acidic and rich in cysteine (SPARC) is an ECM protein with many biological functions that is overexpressed in cirrhotic livers and upregulated in activated hepatic stellate cells (aHSCs). We have recently shown that SPARC downregulation ameliorates liver fibrosis in vivo. To uncover the cellular mechanisms involved, we have specifically knocked down SPARC in two aHSC lines [the CFSC-2G (rat) and the LX-2 (human)] and in primary cultured rat aHSCs. Transient downregulation of SPARC in hepatic stellate cells (HSCs) did not affect their proliferation and had only minor effects on apoptosis. However, SPARC knockdown increased HSC adhesion to fibronectin and significantly decreased their migration toward PDFG-BB and TGF-β(1). Interestingly, TGF-β(1) secretion by HSCs was reduced following SPARC small interfering RNA (siRNA) treatment, and preincubation with TGF-β(1) restored the migratory capacity of SPARC siRNA-treated cells through mechanisms partially independent from TGF-β(1)-mediated induction of SPARC expression; thus SPARC knockdown seems to exert its effects on HSCs partially through modulation of TGF-β(1) expression levels. Importantly, collagen-I mRNA expression was reduced in SPARC siRNA-transfected HSCs. Consistent with previous results, SPARC knockdown in aHSCs was associated with altered F-actin expression patterns and deregulation of key ECM and cell adhesion molecules, i.e., downregulation of N-cadherin and upregulation of E-cadherin. Our data together suggest that the upregulation of SPARC previously reported for aHSCs partially mediates profibrogenic activities of TGF-β(1) and PDGF-BB and identify SPARC as a potential therapeutic target for liver fibrosis.     

Effects of transforming growth factor-beta 1 and fibronectin on SPARC expression in cultures of human periodontal ligament cells

Transforming growth factor-beta(1) (TGF-beta(1)) increases synthesis of secreted protein, acidic and rich in cysteine (SPARC), as well as fibronectin (FN) and type I collagen. However, little is known about the regulatory mechanism of SPARC expression. We examined the effect of FN on SPARC expression by TGF-beta(1) in cultures of human periodontal ligament cells (HPL cells). TGF-beta(1) increased the SPARC and SPARC mRNA levels in HPL cells. Extracellular matrix (ECM) produced by HPL cells in the presence of TGF-beta(1) also increased the SPARC levels. Contents of FN and type I collagen in the ECM were increased by TGF-beta(1). HPL cells cultured on FN-coated plates secreted more SPARC than those on non-coated plates. However, type I collagen had little effect on SPARC levels. The addition of anti-alpha5 antibody to the cultures abolished the increase in SPARC mRNA expression by TGF-beta(1). This study demonstrated that FN may be partly involved in the increase in SPARC expression by TGF-beta(1) in HPL cells.     

Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.     

Expression of SPARC/osteonectin/BM4O in the human gut: predominance in the stroma of the remodeling distal intestine

SPARC is a glycoprotein of the extracellular matrix that exhibits a number of biological functions such as disruption of cell adhesion and modulation of matrix metalloprotease expression. These properties, in concert with the expression of the molecule during development, repair, and neoplastic progression, suggest that SPARC has an important role in remodeling in a variety of tissues. However, the role of SPARC in the intestine is unclear since the development expression and tissular origin of SPARC in this organ appears to be species-dependent. As a first step to investigate the function of SPARC in the tissues of the intestine, we have analyzed its expression at the protein and mRNA levels in the human fetal and adult small intestinal and colonic mucosa as well as in intestinal cell models. Our results show that SPARC expression is differentially regulated during development and along the length of the human intestine. In the colon, SPARC was predominantly found at the epithelial-mesenchymal interface at the fetal stage, below detection levels in the normal adult, but re-expressed in the stroma of colonic tumors. In the small intestine, low levels of SPARC expression were observed at an early stage of morphogenesis (between 9 and 11 weeks) but expression was not detected at subsequent developmental stages nor was it induced in the mucosa of Crohn's disease. While SPARC appeared to be produced mainly by mesenchymal and stromal cells in the intact intestine it was not detected in colon cancer cells. Taken together, these results indicate that SPARC is subject to an onco-fetal pattern of expression in the stroma of the colonic mucosa while its expression is much more restricted in the small intestine, suggesting a differential involvement of this molecule in the extracellular matrix remodeling occurring along the length of the developing and diseased human intestinal mucosa.     

Retention of the matricellular protein SPARC in the endoplasmic reticulum of chondrocytes from patients with pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is an autosomal dominant disease characterized by dwarfism, morphological irregularities of long bones and hips, and early-onset osteoarthritis. This disease has been attributed to mutations in a structural protein of the cartilage extracellular matrix (ECM), cartilage oligomeric matrix protein (COMP), which result in its selective retention in the chondrocyte rough endoplasmic reticulum (ER). Accumulation of excessive amounts of mutated COMP might reflect a defect in protein trafficking by PSACH chondrocytes. Here we identify the matricellular protein SPARC as a component of this trafficking deficit. SPARC was localized to the hypertrophic chondrocytes in the normal human tibial growth plate and in cultured control cartilage nodules. In contrast, concentrated intracellular depots of SPARC were identified in nodules cultured from three PSACH patients with mutations in COMP. The accumulated SPARC was coincident with COMP and with protein disulfide isomerase, a resident chaperone of the rough ER, whereas SPARC and COMP were not coincident in the ECM of control or PSACH nodules. SPARC-null mice develop severe osteopenia and degenerative intervertebral disc disease, and exhibit attenuation of collagenous ECM. The retention of SPARC in the ER of chondrocytes producing mutant COMP indicates a new intracellular function for SPARC in the trafficking/secretion of cartilage ECM.     

Localization of the Extracellular Matrix Protein SC1 Coincides with Synaptogenesis During Rat Postnatal Development

SC1 is an extracellular matrix protein that belongs to the SPARC family of matricellular molecules. This anti-adhesive protein localizes to synapses in the adult rat brain and has been postulated to modulate synapse shape. In this study, increased levels of SC1 were detected from postnatal days 10–20, with a peak at postnatal day 15, a period of intense synaptogenesis. During this time, increased colocalization of SC1 with the synaptic marker synaptophysin was observed in synapse-rich regions of the cerebellum and the cerebral cortex. These findings indicate that the pattern of SC1 localization coincided with synaptogenesis during rat postnatal development.     

Expression and characterization of murine hevin (SC1), a member of the SPARC family of matricellular proteins.

Hevin, also known as SC1, MAST 9, SPARC-like 1, RAGS1 and ECM2, is a member of the SPARC-related family of matricellular proteins. Mouse hevin is 53% identical to mouse SPARC, and both proteins share a follistatin-like module and an extracellular Ca(2+)-binding (E-C) domain. SPARC functions as a modulator of cell-matrix interactions, a regulator of growth factor activity, a de-adhesive protein, and a cell cycle inhibitor. Although the functions of mouse hevin are unknown, its human orthologue has been shown to be de-adhesive for endothelial cells. We now report the production of recombinant mouse hevin in insect cells through the use of a baculoviral expression system and its purification by anion-exchange, size-exclusion chromatography, and isoelectric focusing. Furthermore, we have produced rat anti-hevin monoclonal antibodies (MAbs) that have been characterized by indirect and capture ELISAs, immunoblotting, immunoprecipitation, and immunohistochemistry (IHC). Recombinant hevin, present as a soluble factor or bound to tissue-culture plastic, inhibited the spreading of bovine aortic endothelial cells in vitro. IHC analysis of hevin in normal human and mouse tissues revealed a limited expression pattern in many tissues, with particularly dominant staining in dermis, ducts, vasculature, muscle, and brain. In lung and pancreatic tumor xenografts, we found distinct reactivity with MAbs that were selective for stromal cells, tumor cells, and/or endothelial cells. Although similar to SPARC in its anti-adhesive activities, hevin nevertheless exhibits a distinctive histological distribution that, in certain invasive tumors, is associated with desmoplasia.     

Changes in activity and expression of phosphofructokinase in different rat brain regions after basal forebrain cholinergic lesion

Selective lesion of rat basal forebrain by the cholinergic immunotoxin 192IgG-saporin was used as an animal model to address the question of whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease may be related to impaired cholinergic transmission. At different times after creating the immunolesion, the isoenzyme pattern and steady-state mRNA levels of the key glycolytic enzyme phosphofructokinase were determined in cortex, hippocampus, basal forebrain and nucleus caudatus. The loss of cholinergic input was accompanied by a persistent decrease in choline acetytransferase and acetylcholine esterase activities in the cortical target areas similar to the cholinergic malfunction seen in Alzheimer's dementia. The basal forebrain lesion induced by the immunotoxin resulted in a transient increase in phosphofructokinase activity peaking on day 7 after inducing the lesion in cortical areas. In parallel, an increased steady-state level of phosphofructokinase mRNA was determined by RT/real-time PCR and in situ hybridization. In contrast, analysis by western blotting and quantitative PCR revealed no changes in the phosphofructokinase isoenzyme pattern after immunolesion. It is concluded that common metabolic mechanisms may underlie the degenerative and repair processes in denervated rat brain and in the diseased Alzheimer's brain.     

Altered chemokine expression in the spinal cord and brain contributes to differential interleukin-1beta-induced neutrophil recruitment

The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT-PCR and enzyme-liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin-1beta (IL-1beta) injection resulted in up-regulated chemokine expression in both brain and spinal cord. CINC-3 mRNA was elevated above CINC-1 and CINC-2alpha, with expression levels for each higher in spinal cord than in brain. By ELISA, IL-1beta induced greater CINC-1 and CINC-2alpha expression compared to CINC-3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC-2alpha protein were observed in response to IL-1beta injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC-2beta mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments.     

The TRH neuronal phenotype forms embryonic cell clusters that go on to establish a regionalized cell fate in forebrain

How neurons diversify in developing brain to produce discrete cell fates in their appropriate regions remains a fundamental question. Embryonic Xenopus was previously used to identify juxtaposed embryonic cells that first express proopiomelanocortin mRNA in forebrain and pituitary, supporting the idea that this neuropeptide phenotype is induced locally. (Hayes and Loh, 1990, Development 110:747–757). To begin to examine how a more widespread population of forebrain cells is set up, the present focus is on the thyrotropin-releasing hormone (TRH) phenotype. Serial section in situ hybridization histochemistry produced the unexpected finding that the adult-like TRH system spanning forebrain and comprising over six different telencephalic and diencephalic nuclei, is preceded by an embryonic TRH cell population that is initially localized and then highly regionalized in the area from which the adult pattern develops. Thus, the first TRH cells, detected in vivo after 35 h (stage 29/30), were confined to discrete anterior or posterior bilateral clusters in embryonic forebrain or hindbrain. Thereafter, the TRH cell clusters in diencephelon, but not hindbrain, expanded to form rows, extending anteriorly into telencephalon and bifurcating posteriorly around the infundibulum. By 80 h (stage 42), after extensive brain morphogenesis, these forebrain rows showed regional differences in levels of TRH and mRNA corresponding to the specific brain nuclei that have been shown to contain TRH cells in adult. These findings show that subsets of phenotype-specific forebrain cell first form a regionalized neuronal cell fate before distinct brain nuclei form. This is turn points to the testable hypothesis in Xenopus that certain neuronal cell fates in forebrain may be dictated by cell lineage or local induction. 1994 John Wiley & Sons, Inc.