Clusterin (2. Clusterin regulation)

Both a transient heat shock (20 min at 42 degrees C) and various oxidative stresses, including hydrogen peroxide, superoxide anion, hyperoxia and UVA exposure, induce a strong increase in clusterin mRNA levels as assessed by northern blot. Nuclear run-on analysis suggests that transcriptional activation is involved in inducing clusterin mRNA in response to heat shock. Using pulse-chase analysis of control and heat shocked cells, it is shown that clusterin mRNA is translated and secreted, thus resulting in increased extracellular levels of the protein following heat shock. To investigate the function of clusterin in response to these stresses, clusterin anti-sense transfectants that stably express virtually no clusterin at the mRNA and protein level were generated in A431 cells. These anti-sense transfectants are shown to be highly sensitive to apoptotic cell death induced by heat shock or oxidative stress compared with wild-type A431 cells or control transfectants. Taken together, these results show that clusterin gene expression is induced in response to heat shock and oxidative stress in human A431 cells, and confers cellular protection against heat shock and oxidative stress ^Ref .

Clusterin gene and promoter

the only DNA region strictly conserved between clusterin gene proximal promoters from different vertebrate classes is a 14-bp DNA element which is specifically recognized by the HSF1 (and HSF2169) transcription factor and which can mediate heat-shock-induced transcription in transient expression assays. Conversely, the avian clusterin proximal promoter, point-mutated at the level of this element, no longer transmits heat-shock activation. These findings provide a possible explanation for the high sensitivity of clusterin expression to environmental changes and allow the classification of clusterin as an extracellular version of heat-shock protein ^Ref .
The gene encoding rat clusterin was isolated and characterized, and its cytosine methylation pattern in various tissues was analyzed. Several putative regulatory DNA elements were identified, including a consensus AP-1 site in the 5‘ flanking region. Two AP-1 sites and two transforming growth factor-beta inhibitory elements, one AP-2 site and eight half-sites for glucocorticoid/ androgen response elements were found within the first intron, and one cAMP response element was found in the first exon. The cytosine methylation pattern indicated that testicular or epididymal DNA in the rat is hypomethylated in the region between positions –534 and –99 of the clusterin gene, when compared with tissues with lower levels of expression such as prostate as well as liver, lung, kidney and spleen ^Ref .
Human clusterin gene contains a Myb binding site in its 5‘ flanking region, which interacts with bacterially synthesized B-MYB protein and mediates B-MYB-dependent transactivation of the clusterin promoter in transient transfection assays. Endogenous clusterin expression is induced in mammalian cell lines following transient transfection of a B-MYB cDNA. Blockage of secreted clusterin by a monoclonal antibody results in increased apoptosis of neuroblastoma cells exposed to the chemotherapeutic drug doxorubicin. Thus, activation of clusterin by B-MYB may be an important step in the regulation of apoptosis in normal and diseased cells ^Ref .
The Wnt signaling pathway specifically regulates one out of three clusterin mRNA variants via TCF1. This clusterin transcript is shorter at the 5‘ end than reported by the refseq database, and produces the intracellular 60 kDa CLU protein isoform which is secreted as a ~80 kDa protein after post-translational processing ^Ref .

TGFbeta

TGFbeta stimulates both clusterin mRNA and protein levels, and induces its accumulation in the nucleus of CCL64 cells. A 1.3-kilobase rat clusterin pro-moter/luciferase reporter construct was created and it was demonstrated that TGFbeta enhances luciferase activity 2.5–6-fold in transient transfection assays of epithelial, endothelial, and fibroblast cell lines. Deletional analysis reveals that an AP-1-binding site (5‘-TGAGTCA) in the minimal promoter region is necessary for initiating transactivation by TGFbeta. A single T to G base mutation in the AP-1 site (5‘-TGAGGCA) abolishes TGFbeta-induced clusterin promoter transactivation. In transcription factor decoy experiments, 23-mer oligonucleotides of wild type AP-1 reduce TGFbeta induction of clusterin mRNA levels and promoter transactivation, while an oligonucleotide containing the mutated AP-1 site has no effect. Two specific protein kinase C inhibitors, GF109203X and calphostin C, block TGFbeta-induced clusterin mRNA levels and promoter transactivation. Together these results indicate that TGFbeta regulates clusterin gene expression through an AP-1 site and its cognate transcription factor AP-1, and requires the involvement of protein kinase C ^Ref .

OX-PAPC and Interleukin-6

Treatment of HepG2 cells with oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoryl choline (OX-PAPC), or biologically active lipids present in mildly oxidized low density lipoprotein, increased clusterin, and decreased paraoxonase (PON) mRNA levels. Antibodies to IL-6 blocked these changes. IL-6 treatment in the absence of OXPL produced the same pattern of mRNA changes observed with OXPL treatment alone. In vivo, OX-PAPC injected into C57BL/6J mice resulted in a marked reduction in PON activity and an increase in clusterin levels in plasma after 16 h. Injection of OX-PAPC into IL-6-deficient C57BL/6J mice (IL-6 -/-) did not alter either PON activity or apoJ levels ^Ref .

Glucocorticoids

In the kidney-derived epithelial cell line MDCK, clusterin mRNA is repressed by glucocorticoids and by progesterone. Treatment with epidermal growth factor also represses clusterin gene expression in MDCK cells. Incubation with 12-O-tetradecanoyl-phorbol-13-acetate, which activates protein kinase C (PKC), induces clusterin mRNA, while chelerythrine, an inhibitor of PKC, represses clusterin gene expression, suggesting that the clusterin gene responds to signalling pathways involving PKC ^Ref .

Lactogenic Hormones

The effect of lactogenic hormones and EGF on the expression of involution-induced genes in HC11 mammary epithelial cells was studies. Insulin, dexamethasone, prolactin, and its combinations did not affect expression of the genes. When cells were cultured in growth medium containing EGF the expression of clusterin was strongly inhibited in a dose-and time-dependent manner ^Ref  .

Angiotensin II

Angiotensin II suppression of clusterin expression in the liver of hypertensive rats may represent a specific response to high levels of circulating angiotensin II or a response to hypertensive injury ^Ref .

Testosterone

Testosterone repressed clusterin gene expression is rapidly induced in early involution of the mouse mammary gland, after weaning, and in the rat ventral prostate, after castration. A search for involution-enhanced DnaseI footprints in the proximal mouse clusterin gene promoter led to the identification and characterization (by Dnase I footprinting and EMSA) of a twin nuclear factor 1 (NF1) binding element at –356/-309, relative to the proposed transcription start site; nuclear extracts from 2-day involuting mouse mammary gland showed an enhanced footprint over the proximal NF1 element; extracts from involuting prostate showed enhanced occupancy of both NF1 binding elements. Subsequent EMSA and Western analysis led to the detection of a 74-kDa NF1 protein whose expression is triggered in early involution in the mouse mammary gland; such an induced protein is not found in the involuting rat ventral prostate. This protein was not found in lactation where three other NF1 proteins of 114, 68, and 46 kDa were detected. Reiteration of the epithelial cell apoptosis associated with early mammary gland involution, in vitro, in a primary cell culture system, triggered the appearance of the 74-kDa NF1. Overlaying the cells with laminin-rich extracellular matrix suppressed the apoptosis and the expression of the 74-kDa NF1 and, in the presence of lactogenic hormones, initiated milk protein gene expression and the expression of two of the lactation-associated NF1 proteins (68 and 46 kDa). This study, thus, identifies the occurrence of a switch in expression of different members of the family of NF1 transcription factors as mammary epithelial cells move from the differentiated to the involution/apop­totic state, and it is likely that the involution-specific 74-kDa NF1 accounts for the enhanced NF1 footprint detected on the clusterin promoter with extracts of mouse mammary gland ^Ref .

To investigate whether endocrine disruptors affect spermatogenesis through an clusterin-dependent mechanism, daily oral doses of testosterone (50, 200 and 1,000 microg/kg), flutamide (1, 5 and 25 mg/kg), ketoconazole (0.2, 1, 5 and 25 mg/kg), diethylhexylphtha­late (10, 50 and 250 mg kg), nonylphenol (10, 50, 100 and 250 mg/kg), octylphenol (10, 50 and 250 mg kg), diethylstilbesterol (10, 20 and 40 microg/kg) or corn oil (control) were administered to 5 week-old, male Sprague-Dawley rats for 3 weeks. Following treatment with these endocrine disruptors, testicular expression of clusterin mRNA was analyzed using reverse transcription-polymerase chain reaction. Compared with the control, the lowest dose of testosterone (50 microg/ kg/day) significantly increased expression of clusterin mRNA, whereas 200 and 1,000 microg/kg/day testosterone significantly decreased the expression (P<0.05). Flutamide, ketoconazole, diethylhexylphtha­late, nonylphenol, octylphenol and diethylstilbesterol significantly decreased clusterin mRNA expression in testes at all doses studied, with the exception of 1 mg/kg/day flutamide (P<0.05). These results suggest that endocrine disruptors might decrease spermatogenesis in testes by decreasing expression of clusterin mRNA ^Ref .

Estrogens

Clusterin mRNA expression in rat endometrial adenocarcinoma cells is tightly regulated by estrogens and anti-estrogens in vitro and in vivo, and there is a complex mechanism of regulation of clusterin expression in the normal and cancerous endometrium ^Ref .

Vitamin A

in vitamin A-deficient (VAD) rats before and after administration of all-trans retinoic acid (ATRA), genes, the transcription of which was influenced by ATRA, were isolated. One gene was isolated, the transcription of which was reduced to about 70% by ATRA- clusterin. The effect of ATRA on clusterin expression may be direct, since the promoter of Sgp-2 contains a putative ATRA-responsive element (RARE) ^Ref .

TSH

A prominent secretory glycoprotein was detected in the culture medium of porcine thyrocytes which was identified as clusterin by microsequencing. Treatment of thyrocytes with thyroid stimulating hormone revealed a tight regulation of both synthesis and secretion of clusterin, with a distinct fraction of clusterin being always associated with the cells [[n115]].

Thyroid Hormones

Clusterin was found to be down-regulated by thyroid hormones in HepG2 cell line ^Ref .

Thrombin

Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32–0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C ^Ref .

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