Rat Cystatin Research Articles

Cystain C and neuropeptid Y levels in brain tissues after experimental subarachnoid hemorrhage.

The aim of this study was to investigate the changes in the levels of cystatin C, which protects neurodegeneration in the central nervous system with the inhibition of cysteine protease and by inducing autophagy in the pathogenesis of cerebral vasospasm and levels of vasoconstrictive neuropeptid Y (NPY) in the brain tissue homogenates of rat model of subarachnoid hemorrhage (SAH). Three experimental groups were used: Day 2 and Day 7 groups after SAH, and also a control group. There were seven Wistar albino rats in each group. SAH was accomplished by transclival basilar artery puncture. Rat cystatin C, rat NPY were determined with ELISA in brain tissue homogenates. Day 2 group showed significantly enhanced cystatin C values in comparision with the control group (P=0.048). NPY levels between the Day 2 and Day 7 groups and the control groups were not significantly different (P=0.315). In histopathological examination, there was less neuronal loss in the Day 2 group than in the Day 7 group. Regarding our results, it would be more valuable to measure NPY levels in specific brain areas. The increased cystatin C levels on the second day after SAH is probably a pathophysiologic mechanism to organize protease activity.

Immunofluorescence localization of cystatins in human lacrimal gland and in the exorbital lacrimal gland of the rat.

Cystatins are widely distributed natural inhibitors of cysteine proteinase. They occur both intra and extracellularly in various cells and tissue fluids including tears. Using an immunofluorescence technique with antibodies against rat cystatin S, an inhibitor of submandibular gland origin, cystatin-like immunoreactive material was demonstrated in the acinar cells of the exorbital lacrimal gland of the rat. Administration of the cholinergic agonist carbachol caused a depletion of cystatin from the acinar cells. This depletion was followed by a partial restitution in 6-8 h. Administration of the beta-adrenergic agonist isoproterenol for 4 days, which caused a marked hypertrophy of the submandibular gland, had no effect on the structure, weight, or cystatin content of the exorbital lacrimal gland. After such treatment, however, single large cells with intense staining for cystatin were encountered. Cystatin-like immunoreactive material was also demonstrated in human lacrimal gland using antibodies against human cystatin S. These data suggest the notion that tear cystatins are secreted by the lacrimal glands.

Functional expression of recombinant human stefin A in mammalian and bacterial cells

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-1 monkey kidney cells utilizing pET and recombinant vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS–PAGE and Western blot analysis utilizing a polyclonal antibody against rat cystatin α. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A (∼10 kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BS-C-1 cells, was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells, on the other hand, can provide a significant research tool to study the functional roles of stefin A in mammalian systems such as regulation of cysteine proteases.

Expression of cystatin C prevents oxidative stress-induced death in PC12 cells

Cystatin C, an inhibitor of cysteine proteinases, is suggested to be involved in oxidative stress-induced apoptosis of cultured CNS neurons and various neuronal diseases in vivo; however, little is known about its mechanism of action. To address the role cystatin C plays in oxidative stress-induced neuronal cell death, we established PC12 cell lines that stably expressed rat cystatin C. These cystatin C-expressing PC12 cells showed remarkable resistance to high (50%) oxygen atmosphere. This resistance correlate with expression levels of cystatin C, demonstrating that cystatin C has a protective effect on high oxygen-induced cell death. In contrast, in a normal (20%) oxygen atmosphere neither control nor cystatin C-expressing PC12 cells showed a significant change in the number of living cells, indicating that cystatin C does not play an important role in the regulation of cellular proliferation. Furthermore, the cystatin C-expressing cell line also resisted other oxidative stresses, including glutamate- and 13- l-hydroperoxylinoleic acid (LOOH)-induced cell death. These results demonstrate that cystatin C has protective effects against various oxidative stresses that induce cell death.

Salivary (SD-type) cystatins: over one billion years in the making--but to what purpose?

Human saliva contains relatively abundant proteins that are related ancestrally in sequence to the cystatin superfamily. Most, although not all, members of this superfamily are potent inhibitors of cysteine peptidases. Four related genes have been identified, CST1, 2, 4 and 5, encoding cystatins SN, SA, S, and D, respectively. CST1, 4, and probably CST5 are now known to be expressed in a limited number of other tissues in the body, primarily in exocrine epithelia, and the term SD-type cystatin is more appropriate than 'salivary cystatin'. These genes are co-ordinately regulated in the submandibular gland during post-natal development. The organization of these tissue-specifically-expressed genes in the genome, and their phylogeny, indicate that they evolved from an ancestral housekeeping gene encoding the ubiquitously expressed cystatin C, and are members of a larger protein family. Their relationship to rat cystatin S, a developmentally regulated rodent submandibular gland protein, remains to be established. In this review, the evolution of the SD-type cystatins in the cystatin superfamily, their genomics, expression, and structure-function relationships are examined and compared with known cystatin functions, with the goal of providing clues to their biological roles.

Characterization of a cysteine proteinase inhibitor induced during neuronal cell differentiation.

A rat homolog of human cystatin E/M was identified by differential display of transcripts induced during neuronal cell differentiation. A member of the family 2 cystatins, rat cystatin E/M is secreted, glycosylated and developmentally regulated. Rat cystatin E/M is expressed in brain, and is induced during differentiation of a conditionally immortalized E17 rat hippocampal cell line (H19-7) by bFGF or activated Raf via MEK-dependent and -independent signaling pathways. Rat cystatin E/M protein is increased post-transcriptionally in PC12 cells, and the protein is secreted into the medium of primary embryonal hippocampal cultures. Analysis of the K (i) of recombinant His-tagged rat cystatin E/M toward cathepsins B and H revealed that rat cystatin E/M has an inhibitor profile distinct from that of other members of the cystatin family. Motif swapping between rat cystatin E/M and human cystatin C, a well-characterized cystatin, identified some residues that can contribute to the specificity of inhibition. Taken together, these results describe a member of the cystatin family that has a distinct inhibitor profile and may play a role in neuronal development.

CYSTATIN C IN A RAT MODEL OF END-STAGE RENAL FAILURE

Background: Cystatin C (MW 13 kDa) serum concentration reflects glomerular filtration rate better than creatinine. Like other low-molecular weight proteins it is not eliminated by dialysis. Still, cystatin C serum concentrations do not rise progressively in end-stage renal failure and rarely exceed 10 mg/L (i.e. 8 times the upper limit of normal). Objective: To study cystatin C kinetics in a rat model of end-stage renal failure. Methods: Sequential bilateral nephrectomy was performed seven days apart in 13 male Sprague-Dawley rats as described by Levine and Saltzman. Serum cystatin C (Cystatin C PET-kit, DAKO), creatinine and total protein were measured in daily intervals after the second nephrectomy. Linearity of the anti-human cystatin C assay for rat cystatin C was tested using dilutions of uremic rat serum. Rats were sacrificed for signs of severe uremia on days 10 (n = 5), 11 (n = 4) and 12 (n = 5). Results: At baseline, mean (± SE) cystatin C was 1.59 ± 0.041 mg/L, creatinine 19.6 ± 1.2 μmol/L. Following bilateral nephrectomy, cystatin C immediately rose to 3.82 ± 0.15 mg/L, creatinine to 312 ± 20 μmol/L. During the following days, cystatin C concentration stabilized to 4 mg/L approximately whereas creatinine continued to rise to 822 ± 185 μmol/L on day 12. Correction for the decrease in serum total protein concentration from 48.9 ± 2.3 g/L to 37.4 ± 3.6 g/L did not alter these results. Conclusion: The kinetics of cystatin C and creatinine in this rat model of end-stage renal failure are in accordance withh uman data suggesting a change in cystatin C production or extra-renal elimination in severe chronic uremia.

Involvement of cystatin C in oxidative stress-induced apoptosis of cultured rat CNS neurons

Oxidative stress is involved in neuronal degeneration in cerebrovascular injury, neuropathology and aging. When rat CNS neurons were cultured in a high (50%) oxygen atmosphere, the neurons died. This high oxygen-induced cell death showed features of apoptotic cell death, characterized by DNA fragmentation, and was blocked by inhibitor of protein synthesis. We found that cystatin C and HuC mRNA, the products of which are an inhibitor of cysteine proteases and an RNA binding protein, respectively, were up-regulated in neurons cultured in the high oxygen atmosphere. In the present study, we focused on cystatin C. Cystatin C protein levels were also increased in neurons cultured in the high oxygen atmosphere. In situ hybridization with an RNA probe for rat cystatin C and immunocytochemistry with anti-human cystatin C antibody showed that microtubule-associated protein 2 (MAP2)-positive neurons expressed cystatin C mRNA and protein, respectively, in the high oxygen atmosphere. These results indicated that oxidative stress stimulates an increase in cystatin C expression in cultured neurons, and that cystatin C might have important roles in regulation of apoptosis elicited by oxidative stress.

Autonomic regulation of cystatin S gene expression in rat submandibular glands

Innervation of rat submandibular and parotid glands by the autonomic nervous system regulates saliva volume, its rate of secretion and its composition. The autonomic nervous system also plays a regulatory role in the differentiation and growth of salivary glands, and in the expression of specific sets of genes. Rat cystatin S, a member of family 2 of the cysteine proteinase inhibitor superfamily, is expressed in submandibular and parotid glands of human and rat. In the rat, cystatin S gene expression is tissue- and cell type-specific, is temporally regulated during postnatal development, and not observed in adult animals. The β-adrenergic agonist isoproterenol (IPR) induces hypertrophic and hyperplastic enlargements of rat salivary glands and the expression of a number of genes including cystatin S. Sympathectomy reduces, but does not completely block, IPR-induced expression of the cystatin S gene in submandibular glands of adult female rats, indicating the participation of sympathetic factor(s) in its regulation. Bilateral parasympathectomy also reduces IPR-induced cystatin S gene expression, suggesting a role of the parasympathetic nervous system in its regulation. Experiments described in this paper suggest that similar factor(s) arising from both the sympathetic and parasympathetic branches of the autonomic nervous system simultaneously participate in IPR-induced cystatin S gene expression in submandibular glands.

Molecular cloning, enhancement of expression efficiency and site-directed mutagenesis of rat epidermal cystatin A.

A rat cystatin A cDNA clone was isolated from a lambda ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor. The obtained clone contained a partial coding region of the inhibitor, lacking the 5'-untranslated region and coding sequence for the NH(2)-terminal 13 residues. The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level. To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2. In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0. 5 mg of the purified recombinant protein per 1 liter culture being produced. Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA. The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group. Using the expression system, two kinds of point mutation, the conservative Val54 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain. This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A.

The 5′-Flanking Sequence and Regulatory Elements of the Cystatin S Gene

The gene encoding rat cystatin S (Cys S), a salivary gland-specific secretory protein, has CAAT and TATA boxes upstream of the inititation codon (Cox and Shaw, 1992), and contains regions that resemble those of other hormonally responsive eukaryotic genes. The 5′-flanking sequence of the rat Cys S gene has a potential CREB/AP-1 binding site (Rupp et al., 1990; Trejo et al., 1992), two potential glucocorticoid responsive elements (GREs, Drouin et al., 1989), and a possible GR/PR (glucocorticoid/progesterone) responsive element (Forman and Samuels, 1990). One of these potential GREs is adjacent to a potential AP-2 binding site, and another is typical of the glucocorticoid and progesterone receptor binding site. In this report, we have identified three regions in the 5′-flanking region of the Cys S gene that are found in salivary gland-specific genes (Ting et al., 1992) with a GT-rich region located between conserved elements II and III. Transfection experiments described in this paper suggest that a 281-bp DNA fragment from the Cys S gene promoter region with conserved elements II and III, the GT-rich region, and a possible GR/PR responsive element contains a negative regulatory element. In addition, our experiments suggest that the GT-rich region by itself is acting as a positive regulatory element.

Isoproterenol-induced expression of the cystatin S gene in submandibular glands of parasympathectomized rats

Parasympathetic innervation of rat submandibular and parotid glands regulates saliva volume, its rate of secretion and its composition. It also has a regulatory role in hypertrophy and hyperplasia of salivary glands, and in the expression of specific sets of genes. Rat cystatin S is a member of family 2 of the cysteine proteinase inhibitor superfamily. Cystatin S gene expression is tissue- and cell type-specific, temporally regulated during postnatal development, and not observed in adult animals. Isoproterenol (IPR), a β-adrenergic agonist, induces hypertrophic and hyperplastic enlargement of rat salivary glands and expression of a number of genes including cystatin S. Sympathectomy reduces, but does not completely block IPR-induced expression of the cystatin S gene in the submandibular glands of adult female rats, indicating the participation of sympathetic factor(s) in this regulation. Since both sympathetic and parasympathetic branches of the autonomic nervous system act in parallel in the submandibular gland, it is possible that parasympathetic nerve terminals also provide factor(s) that play a role in regulation of cystatin S gene expression. Experiments described in this paper were designed to test the hypothesis that the parasympathetic nervous system participates in IPR-induced cystatin S gene expression. Bilateral parasympathectomy reduced IPR-induced cystatin S gene expression, suggesting a role of the parasympathetic nervous system in its regulation. Unilateral parasympathectomy in contrast, had no effect on IPR-induced cystatin S gene expression, suggesting that the presence of an intact parasympathetic innervation in the contralateral side permits the `normal' IPR-induced expression of the cystatin S gene in the parasympathectomized gland.

Effect of sympathectomy on isoproterenol-induced expression of the cysteine proteinase inhibitor gene, cystatin S, in rat submandibular glands

The autonomic nervous system regulates the secretory function of salivary glands. The volume, rate of secretion and composition of saliva are regulated by both sympathetic ( α 1-, α 2and β 1-adrenergic) and parasympathetic (muscarinic and cholinergic) receptor systems. The rat cystatin S gene, a member of family 2 of the cysteine proteinase inhibitor superfamily, has a very defined pattern of expression during the postnatal development of the rat submandibular gland. Its expression is not detected in the fetus or in rats up to three weeks of age. After this time, the amount of cystatin S mRNA increases, reaching a conspicuously high concentration at 28 days, and then it declines to a barely detectable level at 32 days of age; cystatin S mRNA is not detectable in the glands of adult animals. However, the β-adrenoreceptor agonist isoproterenol (IPR) induces high concentrations of cystatin S mRNA in the submandibular gland in vivo. This paper reports experiments analysing the participation of the sympathetic nervous system in the IPR-induced expression of the cystatin S gene. Sympathetic denervation (unilateral and bilateral) by removing the superior cervical ganglion 14 days before a single injection of IPR reduced the expression of the cystatin S gene. Chemical denervation by reserpine (a drug that depletes neurotransmitters in sympathetic nerve terminals) also reduced IPR-induced expression of the gene. Morphological analyses of sympathectomized and reserpine-treated glands showed that the structure of the gland was similar to that of glands of intact animals and to those not treated with reserpine. The hypertrophic response to IPR was less obvious in the sympathectomized glands, but was similar in reserpine treated animals. Collectively, these data suggest that even in the presence of a functional β 1-adrenergic receptor pathway, factor(s) from the sympathetic nervous system may be required for IPR-induced expression of the cystatin S gene.

The effect of methotrexate on the expression of a cysteine protease inhibitor (type 2 cystatin) in rat sebaceous glands.

The purpose of this study was to assess whether rat cystatin S, a cysteine proteinase inhibitor, is present in rat sebaceous glands, and to measure the effects of methotrexate on the expression of cystatin in these glands. With methotrexate treatment, the number of skin sebaceous cells expressing cystatin increased from 13.9% to 34.3% (P < .05). A smaller increase (from 15.3% to 23.9%; P = .1) was observed in Zymbal sebaceous glands. Type 2 cystatin could not be detected in the major salivary glands, nor in trachea, lung, stomach, small intestine, large intestine, spleen, liver, kidney, or pancreas, in any of the rats given either saline or methotrexate. Our results suggest that type 2 cystatin is a constituent of normal sebaceous glands, and that the amount of cystatin present in these glands increases with methotrexate administration. We speculate that, in addition to the protective functions ascribed to sebaceous lipids, sebum may augment the physical barrier of skin through secretion of cysteine proteinases that may be pharmacologically modulated.

Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution

Recombinant mouse ( Mus musculus) and rat ( Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized. The mouse and rat inhibitors were both fully active in titrations of papain. Determination of equilibrium constants for dissociation ( K i ) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C ( K i 0.07−0.13 nM). Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors. Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay. The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates. All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues. For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content. In the mouse, a notable high cystatin C content in parotid gland tissue was observed. The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that: a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy.

Up-regulation of cystatin C by microglia in the rat facial nucleus following axotomy.

Cystatin C, a cysteine proteinase inhibitor, is expressed in the central nervous system (CNS) as well as many other organs of mammals. However, little is known concerning whether its expression is regulated under pathological conditions of the CNS and what types of cells are responsible for this regulation. We performed differential hybridization screening of cDNA libraries derived from the rat facial nucleus and found a cDNA of rat cystatin C to be up-regulated following facial nerve axotomy. In situ hybridization using an RNA probe for rat cystatin C revealed that cystatin C mRNA in the facial nucleus was markedly increased in amount by day 7 after axotomy and was then decreased to the normal level by day 50. The intense signal for cystatin C mRNA in the damaged facial nucleus was localized in the glial cells which had the morphological characteristics of microglia. Light and electron microscopic immunohistochemistry using a rabbit antibody specific for cystatin C confirmed that microglia in the damaged facial nucleus were strongly positive for cystatin C. The immunoreactivity was also found in the extracellular space, consistent with the fact that cells producing cystatin C generally secrete this protein. These results demonstrate that cystatin C is markedly up-regulated by microglia in response to axotomy and is probably secreted by these cells into the extracellular space, suggesting that this proteinase inhibitor has (a) significant function(s) in the processes of neuronal degeneration, regeneration, and/or repair subsequent to axotomy.

Expression of a functional rat salivary cystatin S polypeptide in Escherichia coli

Cystatin S is a cysteine proteinase inhibitor that is transiently expressed during rat submandibular gland development and can be induced by isoproterenol in the adult. A cDNA for rat cystatin S which included the entire coding sequence of the secreted cystatin was cloned. A coding region of the cystatin gene was amplified by polymerase chain reaction and cloned into the pGEX-2T expression vector. The chimeric plasmid was transformed into Escherichia coli, and protein expression was induced by isopropyl-ß- d-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and fast protein liquid chromotography on a MonoQ column. The purified recombinant cystatin reacted with antibodies to cystatin S purified from rat submandibular glands and showed an amino-terminal amino acid sequence identical to that of rat cystatin S. The recombinant protein exhibited papain inhibition activity comparable to natural cystatin. This was a successful expression and purification of a functionally and immunologically reactive recombinant cystatin from E. coli, an approach which will be used later towards generating recombinant variants to study the binding and functional domains of this cysteine protease inhibitor.

Bactericidal effect of rat cystatin S on an oral bacterium Porphyromonas gingivalis

We tested antibacterial and antiviral activities of rat cystatin S, a cysteine proteinase inhibitor, belonging to the family 2 cystatins against 18 different bacterial species and poliovirus type 1 (Sabin). Rat cystatin S specifically inhibited the growth of a human oral anaerobic bacterium Porphyromonas gingivalis due to a bactericidal effect.

Expressions of the genes for cysteine proteinase inhibitors cystatin C and cystatin S in rat submandibular salivary gland

Rat cystatin S and rat cystatin C are members of family 2 (cystatin) of the cystatin superfamily. All members of the cystatin family inhibit cysteine proteinases to varying degree. The expression of these two inhibitors, which have a 48% similarity at the nucleotide level, was studied in the submandibular gland using reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot hybridization and in situ hybridization with digoxigenin-Iabelled DNA probes. Both inhibitors were expressed in the serous acinar cells of the submandibular gland. In accord with previous findings, cystatin S mRNA was induced by the β-adrenergic agonist isoproterenol. The level of cystatin S mRNA, which was very low in the glands of untreated rats and was demonstrable by RT-PCR but not by Northern blot hybridization, was not altered by acute inflammation produced by turpentine. Neither the administration of isoproterenol nor acute inflammation had any effect on the level of cystatin C mRNA, indicating that β-adrenoreceptors are not involved in the regulation of the cystatin C gene(s) in the submandibular gland. The data indicate that these two closely related genes, expressed in the same cells, are differently regulated. The consequence of this difference in gene regulation on the physiological and pathological roles of these inhibitors remains to be established.

Induction of synthesis of the rat cystatin S protein by the submandibular gland during the acute phase of experimental Chagas disease

Rats experimentally infected with Trypanosoma cruzi Y strain exhibited hypertrophy of the submandibular gland at 18 days after infection. SDS-PAGE of infected rats saliva revealed the presence of an additional band with an apparent molecular weight of about 13KDa. Electrophoresis of protein saliva and immunochemical analysis with antibody against rat cystatin S confirmed that the protein was identical to that induced by beta adrenergic stimulation.