Hepatocyte-specific clusterin overexpression attenuates diet-induced nonalcoholic steatohepatitis.

Clusterin is a multifunctional glycoprotein that plays important roles and is up-regulated in liver diseases such as hepatitis and hepatocellular carcinoma. However, little is known about the significance of clusterin in the pathogenesis of non-alcoholic steatohepatitis (NASH). The aim of this study is to examine the role of clusterin in progression of steatohepatitis in mice fed a methionine and choline deficient (MCD) diet. We generated hepatocyte-specific clusterin overexpression (hCLU-tg) mice, and hCLU-tg mice showed lower levels of hepatic triglycerides, less infiltration of macrophages and reduction of TNF-α, activation of Nrf-2 than wild-type littermates fed the MCD diet. Also, sustained clusterin expression in liver ameliorated hepatic fibrogenesis by reducing the activation of hepatic stellate cells by MCD diet. Sustained expression of clusterin in liver functioned as a preconditioning stimulus and prevented MCD diet-induced severe steatohepatitis injury via Nrf2 activation. These results demonstrate a novel function of clusterin as an immune preconditioning regulator in various inflammatory diseases including steatohepatitis.

Toll-like receptor 4 signaling is required for clusterin-induced tumor necrosis factor-α secretion in macrophage.

Clusterin is a secretory glycoprotein that is up-regulated in areas of inflammation and under increased levels of oxidative stress. Previously, we demonstrated that clusterin activates NF-κB, and up-regulates the expression of MMP-9 and TNF-α. In this research, we extend our previous findings by reporting that such clusterin-induced macrophage response is mediated via TLR4 signaling. Specifically, we found that TNF-α induced by clusterin was significantly abrogated by pretreatment of TLR4-signaling inhibitors and anti-TLR4 neutralizing antibody. Additionally, a primary culture of macrophages derived from TLR4-signal defective and knockout mice were unresponsive to clusterin, resulting in no TNF-α secretion, whereas macrophages carrying wild-type TLR4 responded to clusterin and induced TNF-α. Moreover, clusterin increased NF-κB promoter activity in HEK-Blue hTLR4 cells, but not in HEK-Blue Null2 cells. To confirm that clusterin elicits TLR4 signal transduction, recombinant clusterin was generated and purified from cell culture. Interestingly, we found that the recombinant clusterin with C-terminal HA-tag induces TNF-α secretion at a significantly lower level compared to an intact form of clusterin without C-terminal HA-tag. Removal of HA-tag from the recombinant clusterin restored its activity, suggesting that C-terminal HA-tag partially masks the domain involved in TLR4 signaling. Furthermore, clusterin enhanced TLR4 mobilization into lipid raft of plasma membrane, and TNF-α and MMP-9 secretion stimulated by clusterin was diminished by pretreatment with methyl-ß-cyclodextrin (MßCD), which was used to disrupt lipid raft. In conclusion, clusterin-induced TNF-α and MMP-9 up-regulation is most likely mediated via TLR4 recruitment into lipid rafts, and these data describe a novel role of clusterin as an endogenous regulator for TLR4 signaling.

Clusterin stimulates the chemotactic migration of macrophages through a pertussis toxin sensitive G-protein-coupled receptor and Gßγ-dependent pathways.

Clusterin induces the expression of various chemotactic cytokines including tumor necrosis factor-α (TNF-α) in macrophages and is involved in the cell migration. According to the results of this study, clusterin induced the directional migration (chemotaxis) of macrophages based on a checkerboard analysis. The chemotactic activity of clusterin was prevented by pretreatment with pertussis toxin (PTX), indicating that the Gαi/o-protein coupled receptor (GPCR) was involved in the chemotactic response of clusterin. Clusterin-stimulated chemotaxis was abrogated in a dose-dependent manner by pretreatment with gallein (a Gßγ inhibitor), indicating the involvement of Gßγ released from the GPCR. In addition, inhibitors of phospholipase C (PLC, U73122) and phosphoinositide 3-kinase (PI3K, LY294002), the key targets of Gßγ binding and activation, suppressed chemotactic migration by clusterin. The phosphorylation of Akt induced by clusterin was blocked by pretreatment with gallein or LY294002 but not with U73122, indicating that Gßγ released from the PTX-sensitive Gi protein complex activated PLC and PI3K/Akt signaling pathways separately. The activation of cellular MAP kinases was essential in that their inhibitors blocked clusterin-induced chemotaxis, and Gßγ was required for the activation of MAP kinases because gallein reduced their phosphorylations induced by clusterin. In addition, the inflammation-induced migration of macrophages was greatly reduced in clusterin-deficient mice based on a thioglycollate-induced peritonitis model system. These results suggest that clusterin stimulates the chemotactic migration of macrophages through a PTX-sensitive GPCR and Gßγ-dependent pathways and describe a novel role of clusterin as a chemoattractant of monocytes/macrophages, suggesting that clusterin may serve as a molecular bridge between inflammation and its remodeling of related tissue by recruiting immune cells.

Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation.

Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU(-/-) mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

Clusterin attenuates the development of renal fibrosis.

Upregulation of clusterin occurs in several renal diseases and models of nephrotoxicity, but whether this promotes injury or is a protective reaction to injury is unknown. Here, in the mouse unilateral ureteral obstruction model, obstruction markedly increased the expression of clusterin, plasminogen activator inhibitor-1 (PAI-1), type I collagen, and fibronectin. Compared with wild-type mice, clusterin-deficient mice exhibited higher levels of PAI-1, type I collagen, and fibronectin and accelerated renal fibrosis in response to obstruction. In cultured rat tubular epithelium-like cells, adenovirus-mediated overexpression of clusterin inhibited the expression of TGF-ß-stimulated PAI-1, type I collagen, and fibronectin. Clusterin inhibited TGF-ß-stimulated Smad3 activity via inhibition of Smad3 phosphorylation and its nuclear translocation. Moreover, intrarenal delivery of adenovirus-expressing clusterin upregulated expression of clusterin in tubular epithelium-like cells and attenuated obstruction-induced renal fibrosis. In conclusion, clusterin attenuates renal fibrosis in obstructive nephropathy. These results suggest that upregulation of clusterin during renal injury is a protective response against the development of renal fibrosis.

Clusterin induces the secretion of TNF-α and the chemotactic migration of macrophages.

Tumor associated macrophages are known to be closely linked with tumor progression and metastasis. On the other hand, clusterin is overexpressed in several tumor types and regarded as a putative tumor-promoting factor due to this overexpression and the subsequent induction of chemoresistance. In our previous study, clusterin was found to induce the expression of matrix metalloproteinase-9 (MMP-9) in macrophages, and MMP-9 is known to be essential for tumor cell migration and invasion via basement membrane breakdown. Because paracrine interactions between tumor cells and surrounding macrophages regulate metastasis, these findings raise the possibility that clusterin promotes the secretion of cytokines in macrophages in addition to MMP-9. Here, we demonstrate that clusterin upregulates the expressions of chemotactic cytokines, that is, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1ß (MIP-1ß), regulated upon activation, normal T cell expressed and secreted (RANTES), and tumor necrosis factor-α (TNF-α) in Raw264.7 macrophages. In particular, clusterin stimulated TNF-α secretion via the activations of ERK, JNK, and PI3K/Akt pathways in a time and dose-dependent manner. Furthermore, clusterin-induced TNF-α secretion was found to play a critical role in the chemotactic migration of Raw264.7 macrophages. It was also found that clusterin acts directly as a chemoattractant for macrophages. Together, these results suggest that clusterin stimulates the expression and secretion of TNF-α, which plays a critical role in promoting macrophage chemotaxis, via ERK, JNK, and PI3K/Akt pathways. Collectively, these findings describe a novel function for clusterin as an inducer of TNF-α in macrophages and their chemotactic migration, and suggest that clusterin has a tumor-promoting effect.

Over-expression of human clusterin increases stress resistance and extends lifespan in Drosophila melanogaster.

Clusterin is a disulfide-linked heterodimeric glycoprotein that has been implicated in a variety of biological processes. Its expression has been shown to be elevated during cellular senescence and normal aging, but it is uncertain whether clusterin protects against aging or whether its expression is a consequence of aging. To investigate the functions of clusterin during organismal aging, we established transgenic Drosophila alleles to induce the expression of the secretory form of human clusterin (hClu(S)) using the Gal4/UAS system. hClu(S) protein (~60 kDa) was detected in both adult homogenates and larval hemolymphs of flies ubiquitously overexpressing hClu(S) (da-Gal4>UAS-hClu(S)) and in motoneurons (D42-Gal4>UAS-hClu(S)). Interestingly, the mean lifespans of these hClu(S)-overexpressing flies were significantly greater than those of control flies that exhibited no hClu(S) induction. hClu(S)-overexpressing flies also showed significantly greater tolerance to heat shock, wet starvation, and oxidative stress. Furthermore, amounts of reactive oxygen species (ROS) in whole bodies were significantly lower in hClu(S)-overexpressing flies. In addition, clusterin was found to prevent the inactivation of glutamine synthetase (GS) by metal-catalyzed oxidation (MCO) in vitro, and this protection was only supported by thiol-reducing equivalents, such as, DTT or GSH, and not by ascorbate (a non-thiol MCO system). Furthermore, this protection against GS inactivation by clusterin was abolished by reacting clusterin with N-ethylmaleimide, a sulfhydryl group-modifying agent. Taken together, these results suggest that a disulfide-linked form of clusterin functions as an antioxidant protein via its cysteine sulfhydryl groups to reduce ROS levels and delay the organismal aging in fruit flies.

Essential role of clusterin in pancreas regeneration.

Based on our previous observations that clusterin induction accompanies pancreas regeneration in the rat, we sought to determine if regeneration might be impaired in mice that lacked clusterin. We studied the impact of absent clusterin on morphogenic and functional features of regenerating pancreas. Clusterin induction was accompanied in the regenerating pancreas by a robust development of new lobules with ductules, acini, and endocrine islets in wild type after partial pancreatectomy. In clusterin knock-out mice, however, pancreatectomy resulted in a poor formation of regenerating lobule. In particular, regeneration of beta-cells was also significantly reduced and was associated with persistent hyperglycemia. Duct cells obtained from pancreatectomized clusterin knock-out mice exhibited impaired beta-cell formation in vitro; this was restored by administration of exogenous clusterin. We suggest that clusterin plays a critical role to promote both exocrine and endocrine regeneration following pancreas injury, as well as for in vitro beta-cell regeneration.

Protective role of clusterin/apolipoprotein J against neointimal hyperplasia via antiproliferative effect on vascular smooth muscle cells and cytoprotective effect on endothelial cells.

OBJECTIVE: Clusterin is induced in vascular smooth muscle cells (VSMCs) during atherosclerosis and injury-induced neointimal hyperplasia. However, its functional roles in VSMCs and endothelial cells remain controversial and elusive. This study was undertaken to clarify the role of clusterin in neointimal hyperplasia and elucidate its mechanism of action. METHODS AND RESULTS: Adenovirus-mediated overexpression of clusterin (Ad-Clu) repressed TNF-alpha-stimulated expression of MCP-1, fractalkine, ICAM-1, VCAM-1, and MMP-9, leading to inhibition of VSMC migration. Both Ad-Clu and secreted clusterin suppressed VSMC proliferation by inhibiting DNA synthesis, but not by inducing apoptosis. Ad-Clu upregulated p53 and p21(cip1/waf1) but downregulated cyclins D and E, leading to suppression of pRb phosphorylation and subsequent induction of G1 arrest in VSMCs. Clusterin deficiency augmented VSMC proliferation in vitro and accelerated neointimal hyperplasia in vivo, but concomitantly impaired reendothelialization in wire-injured murine femoral arteries. Moreover, Ad-Clu significantly reduced neointimal thickening in balloon-injured rat carotid arteries. Clusterin also diminished TNF-alpha-induced apoptosis of human umbilical vein endothelial cells and restored endothelial nitric oxide synthase expression suppressed by TNF-alpha. CONCLUSIONS: These results suggest that upregulation of clusterin during vascular injury may be a protective response against, rather than a causative response to, the development of neointimal hyperplasia.

Clusterin overexpression protects against western diet-induced obesity and NAFLD.

Obesity is a significant risk factor for various metabolic diseases and is closely related to non-alcoholic fatty liver disease (NAFLD) characterized by inflammation and oxidative stress. Clusterin is a multi-functional protein that is up-regulated in the pathogenesis of various metabolic diseases, including obesity and NAFLD. Our previous studies indicated that hepatocyte-specific overexpression of clusterin alleviates methionine choline-deficient (MCD) diet-induced non-alcoholic steatohepatitis (NASH) by activating nuclear factor erythroid 2-related factor 2 (Nrf2). Here we generated transgenic mice with whole-body clusterin overexpression (wCLU-tg) and investigated the role of clusterin in Western diet-induced obesity and NAFLD. We confirmed that obesity parameters and the spectrum of NAFLD of wCLU-tg mice were improved compared to wild type mice. Contrarily, clusterin deficiency deteriorated metabolic disruptions. We also found that clusterin activates target molecules for obesity and NAFLD, namely Nrf2 and AMPK, suggesting that clusterin protects against Western diet-induced obesity and NAFLD by activating Nrf2 and AMPK.

Comparative study on high fat diet-induced 4-hydroxy-2E-nonenal adducts in the hippocampal CA1 region of C57BL/6N and C3H/HeN mice.

In the present study, we investigated the influences of a high fat diet (HD) fed for 12 weeks, on lipid peroxidation and antioxidant enzyme using 4-hydroxy-2E-nonenal (HNE)-modified proteins (HNE-mp) and Cu,Zn-superoxide dismutase (SOD1) in the hippocampal CA1 region (CA1) in C57BL/6N and C3H/HeN mice. Body weights and body weight gains were significantly higher in HD fed C57BL/6N mice than in low fat diet (LD) fed C57BL/6N and LD or HD fed C3H/HeN mice. In the HD fed C57BL/6N and C3H/HeN mice, HNE-mp immunoreactivity and protein levels were much higher than in the LD fed C57BL/6N or C3H/HeN mice. In particular, HNE-mp immunoreactivity and protein levels in HD fed C57BL/6N mice was higher than that in the HD fed C3H/HeN mice. SOD1 immunoreaction was detected in the non-pyramidal cells of C57BL/6N mice, while in the C3H/HeN mice SOD1 immunoreaction was observed in CA1 pyramidal cells. The SOD1 immunoreactivity in the LD fed C57BL/6N and C3H/HeN mice was slightly, but not significantly decreased compared to that in the HD fed C57BL/6N and C3H/HeN mice, respectively. In addition, ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactive microglia in the HD fed C57BL/6N showed hypertrophy of cytoplasm, which is the characteristics of activated microglia. These results suggest that HD fed C57BL/6N mice are more susceptible to lipid peroxidation in the CA1 than in LD fed C57BL/6N and LD or HD fed C3H/HeN mice without any differences of SOD1 expression.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces calcium influx through T-type calcium channel and enhances lysosomal exocytosis and insulin secretion in INS-1 cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been associated with diabetes in several epidemiological studies. However, the diabetogenic action of TCDD on pancreatic cells is unclear. Here, we investigated the direct toxic effects of TCDD on a rat insulin-secreting beta cell line. We found that TCDD enhances exocytosis of MTT formazan and lysosomal proteins such as beta-hexosaminindase and Lamp-1. This TCDD-induced exocytosis was abrogated by T-type calcium channel blockers (mibefradil, flunarizine) but not by an aryl hydrocarbon receptor antagonist (alpha-naphtoflavone). Indeed, cytosolic calcium levels were increased by TCDD. Furthermore, TCDD stimulated insulin secretion, which was inhibited by flunarizine. Taken together, our results suggest that TCDD-induced calcium influx via T-type channels regulates vesicular trafficking, such as lysosomal and secretory granule exocytosis, and that TCDD might exert adverse effects on beta cells by continuous insulin release followed by beta cell exhaustion. This could contribute to the link between TCDD exposure and the risk of developing diabetes.

Arginine deiminase enhances MCF-7 cell radiosensitivity by inducing changes in the expression of cell cycle-related proteins.

After successful clinical application, arginine deiminase (ADI) has been proposed to be a new cancer therapeutic. In the present study, we examined the effect of ADI in combination with ionizing radiation (IR) on MCF-7 cell growth and clonogenic cell death. Cell growth was inhibited by IR in a dose-dependent manner and ADI enhanced the radiosensitivity. ADI itself did not suppress the growth of MCF-7 cells due to the high level of expression of argininosuccinate synthetase (ASS), which convert citrulline, a product of arginine degradation by ADI, to arginine. Previously, it was suggested that ammonia, another product of arginine degradation by ADI, is the main cause of the growth inhibition of irradiated hepatoma cells contaminated with ADI-expressing mycoplasma [van Rijn et al. (2003)]. However, we found that ammonia is not the only factor that enhances radiosensitivity, as enhancement was also observed in the absence of ammonia. In order to identify the enhancing effect, levels of ASS and proteins related to the cell cycle were examined. ASS was unchanged by ADI plus IR, but p21 (a CDK inhibitor) was upregulated and c-Myc downregulated. These findings indicate that changes in the expressions of cell cycle proteins are involved in the enhancement of radiosensitivity by ADI. We suggest that ADI is a potential adjunct to cancer therapy.

Gamma-irradiation enhances RECK protein levels in Panc-1 pancreatic cancer cells.

Radiotherapy is an important treatment for many malignant tumors, but there are recent reports that radiation may increase the malignancy of cancer cells by stimulating expression of type IV collagenases. In this study, we examined changes in matrix metalloproteinase (MMP) inhibitors, such as the tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and RECK, in response to irradiation in Panc-1 pancreatic cancer cells. Irradiation increased RECK protein levels but not mRNA levels, whereas no significant changes were found in TIMP-1 and TIMP-2. The enhanced RECK protein levels were associated with an increase in MMP inhibitory activity. However, irradiation slightly but reproducibly increased the invasiveness of the Panc-1 cells. Like irradiation, treatment of Panc-1 cells with transforming growth factor (TGF)-Beta1 led to a 2-fold increase in RECK protein levels. Transient transfection with Smad3 also increased RECK protein levels, but transfection with Smad7 markedly reduced them. Stable expression of Smad7 and treatment with SB431542, an inhibitor of TGF-Beta receptor I kinase, abolished TGF-Beta1- and radiation-mediated effects on RECK. Furthermore, irradiation increased levels of phosphorylated Smad3. We conclude that radiation post-transciptionally enhances RECK protein levels in Panc-1 cells, at least in part, via TGF-Beta signaling, and that irradiation increases Panc-1 invasiveness via a mechanism that may not be linked to MMP-2 activity.

SIRT1 promotes DNA repair activity and deacetylation of Ku70.

Human SIRT1 controls various physiological responses including cell fate, stress, and aging, through deacetylation of its specific substrate protein. In processing DNA damage signaling, SIRT1 attenuates a cellular apoptotic response by deacetylation of p53 tumor suppressor. The present study shows that, upon exposure to radiation, SIRT1 could enhance DNA repair capacity and deacetylation of repair protein Ku70. Ectopically over-expressed SIRT1 resulted in the increase of repair of DNA strand breakages produced by radiation. On the other hand, repression of endogenous SIRT1 expression by SIRT1 siRNA led to the decrease of this repair activity, indicating that SIRT1 can regulate DNA repair capacity of cells with DNA strand breaks. In addition, we found that SIRT1 physically complexed with repair protein Ku70, leading to subsequent deacetylation. The dominant-negative SIRT1, a catalytically inactive form, did not induce deacetylation of Ku70 protein as well as increase of DNA repair capacity. These observations suggest that SIRT1 modulates DNA repair activity, which could be regulated by the acetylation status of repair protein Ku70 following DNA damage.

Anti-tumor activity of arginine deiminase via arginine deprivation in retinoblastoma.

In spite of recent advances in the treatment of retinoblastoma, chemotherapy is still challenging in high-stage intraocular retinoblastoma or metastatic retinoblastoma. Here, we investigated whether arginine deprivation via arginine deiminase (ADI) could be a new anti-tumor therapy in retinoblastoma cells. Expression of argininosuccinate synthetase (ASS) was detected in human retinoblastoma tissues. Even with a high expression of ASS, ADI effectively inhibited the proliferation of retinoblastoma cells and induced retinoblastoma cell death in a dose-dependent manner. These results indicate that arginine deprivation via ADI could be another treatment option for retinoblastoma due to low ASS activity in retinoblastoma cells.

Functional association of the morphogenic factors with the clusterin for the pancreatic beta-cell differentiation.

Several differentiation or morphogenic factors have known to be involved in the developmental process of endocrine pancreas. However, mechanism of action and functional relation of these molecules are not well elucidated particularly in beta-cell formation from adult pancreatic stem cells. We hypothesized that adult pancreatic stem cells could be activated by the functional resumption of the morphogenic factors that were involved in embryonic development of pancreas in the duct system under the specific conditions such as tissue injuries. Besides the well-established genes including Pdx-1 and Ngn-3, we propose the nestin and clusterin as the new morphogenic factors for beta-cell neogenesis and their functional associations. We found extensive in vivo formation of ductules showing a higher replicating ability following the experimental tissue injury. These neogenic ductules were lined with low epithelial cells positive for the nestin, which has been known as neuronal stem cell marker. In in vitro culture, the nestin-rich epithelial cells of the neogenic ductules also displayed extensive self-replication leading to monolayer of epithelial cell explants and transformed into the insulin secreting beta cells as well as duct cells. Thus, we depicted them as nestin-positive duct stem (NPDS) cells. We found a neogenesis specific protein 'clusterin' in the regenerating pancreatic tissues with concomitant increase of Pdx-1 and Ngn-3 expression. The protein is expressed predominantly in the neogenic pancreas undergoing differentiation. In vitro over-expression of the clusterin gene strongly induces beta-cell transformation from neogenic ductal cells. Insulin expression, both insulin mRNA and peptide levels, was increased and showed glucose dependent manner by ectopic expression of clusterin upon the culture of neogenic ductules when compared to the mock-transfected control, implying that the duct cells transformed functional beta cells. We observed that clusterin over-expression led to up-regulation of Pdx-1 and Ngn-3, and clusterin levels were increased upon the transfection of cDNAs of Pdx-1 or Ngn-3, suggesting a close functional association of these morphogenic factors. In conclusion, we suggest that adult pancreatic stem cells can be recapitulated for neogenesis of insulin secreting beta cells not only by reactivation Pdx-1 and Ngn-3, the classical differentiation factors for pancreas development, but also by the intervention of new morphogenic factors including nestin and clusterin. In particular, by modulation of Pdx-1 and Ngn-3, clusterin induces remarkable differentiation of the functional beta cells secreting insulin in response to glucose stimulation.

The role of clusterin in retinal development and free radical damage.

AIM: To assess the role of clusterin in retinal vascular development and in free radical damage in vivo and in vitro. METHODS: The expression of clusterin, von Willebrand factor (vWF), flk-1, heat shock protein 27 (Hsp27) and heat shock protein 70 (Hsp70) was examined in the retinas of developing mice and oxygen-induced retinopathy (OIR) mice by immunofluorescence staining and western blot analysis. Hydrogen peroxide (H(2)O(2))-pretreated human retinal endothelial cells (HREC) and astrocytes were cultured in the presence or absence of exogenous clusterin, and then the cell viability was measured using the MTT assay and DAPI staining. RESULTS: Clusterin was expressed mainly in the inner retina and co-localised with vWF, an endothelial cell marker. During the mouse developmental process, clusterin expression was decreased, which was similar to the expression of flk-1, vWF and Hsp27. Furthermore, in the OIR model, clusterin expression changed in a similar way to both vWF and Hsp27. Under hypoxic conditions, clusterin expression increased in HREC and astrocytes. In H(2)O(2)-pretreated HREC and astrocytes, clusterin protected against apoptotic cell death. CONCLUSIONS: These results suggest that clusterin is associated with protection from apoptotic retinal cell death in retinal development and in free radical damage.

The role of clusterin in in vitro ischemia of human retinal endothelial cells.

Clusterin has been reported to be up-regulated in diverse pathophysiological stresses, but its role is controversial. In this study, we investigated the role of clusterin under in vitro ischemia of human retinal endothelial cells (HRECs). When HRECs were exposed to oxygen-glucose deprivation (OGD), clusterin expression increased, whereas von Willebrand factor (vWF), occludin, and zonula occludens (ZO-1) markedly decreased. Interestingly, loss of tight junction proteins and death of HRECs in OGD conditions were restored by clusterin treatment. Our results suggest that the enhanced clusterin in OGD conditions may play a protective role against ischemia-induced tight junction protein loss and HRECs death.

Multimerization of expressed protein-arginine methyltransferases during the growth and differentiation of rat liver.

Protein-arginine methylation is a posttranslational modification which yields monomethyl and dimethyl (asymmetric or symmetric) arginines in proteins. We investigated the expressions of PRMT1 and PRMT5 in relation to their catalytic activities in rat liver during growth and differentiation as well as in the pancreas. Western immunoblot analysis revealed that both PRMT1 and PRMT5 proteins were expressed in the cytosol of liver and pancreas with molecular mass of about 42 kDa and 72 kDa, respectively. However, on molecular sieve chromatography, the enzyme activities were eluted at about 500 kDa for PRMT5 and 440 kDa for PRMT1, indicating that the multimer complex of these expressed monomers were catalytically active. While the 500 kDa complex methylated predominantly myelin basic protein (MBP), the 440 kDa complex methylated hnRNP A1 protein. In fetal rat liver, the amount of expressed 42 kDa PRMT1 protein and the enzyme activity to methylate hnRNPA1 protein were 2- to 3-fold and 4- to 5-fold higher, respectively, than those of post-natal livers. While the 72 kDa PRMT5 protein was consistently expressed, its activity varied only about 2-fold. However, PRMT5 to methylate MBP showed one distinct peak at around the 20th day post-natal. Furthermore, while the PRMT1 enzyme activity increased more than 10-fold after 3 days of 70% partial hepatectomy, the amount of expressed PRMT1 protein was only about 3.2-fold higher than the control livers. In summary, we observed that PRMTs are catalytically active only in the form of multimers, but not as a dimer or tetramer of the expressed subunit. Furthermore, the amount of expressed PRMT protein, determined by Western immunoblot, did not correlate with the amount of their catalytic activity, and thus, some uncharacterized additional factor(s) may multimerize PRMTs to express catalytic activities in vivo.