Molecular cloning, expression profile and functional implications of clusterin in the pituitary gland of helmeted guinea fowl (Numida meleagris)

Clusterin is shown to contain putative amphipathic alpha-helices that mediate hydrophobic interactions with numerous types of molecules and may be involved in clearance of cellular debris caused by cell injury or death. To assess this function in vivo, we have cloned the full-length cDNA encoding guinea fowl (Numida meleagris) clusterin and studied its synthesis and expression pattern in specific cell types in pituitary. Quantity of clusterin mRNA expressed in pituitary and endocrine tissues was quantified by real-time PCR. Highest levels were detected in gonads. In situ hybridization showed clusterin mRNA in endocrine cells and folliculostellate cells. Clusterin protein detected by immunohistochemistry was observed in endocrine cells, folliculostellate cells and in colloid. The expression pattern suggests that clusterin is produced by endocrine cells for cytoprotection. Degenerating endocrine cells are phagocytosed by folliculostellate cells and digested by their lysosomal enzymes. In folliculostellate cells clusterin interacts and aggregates with by-products of digestion that subsequently become stored in colloid.     

Are folliculo-stellate cells in the anterior pituitary gland supportive cells or organ-specific stem cells?

Folliculo-stellate cells (FS-cells) in the anterior pituitary gland are star-shaped cells and form tiny follicles. FS-cells are positive for S-100 protein and produce many cytokines or growth factors, such as interleukin-6 (IL-6), leukemia inhibitory factor (LIF), basic fibroblastic growth factor (bFGF) and vascular endothelial cell growth factor (VEGF). Therefore, it is generally accepted that FS-cells regulate endocrine cells through these growth factors. FS-cells also exhibit a phagocytotic activity and are known to work as scavenger cells. In addition to these functions, FS-cells are considered to have some unknown functions. In order to reveal the biological significance of FS-cells in the anterior pituitary gland, we performed a morphological study and obtained some new findings. First, we were interested in the colloid formation in the senescent porcine pituitary gland. We analyzed the colloids and found that clusterin is a major protein in them. We also found that the accumulation of clusterin in the colloids is related to the phagocytotic activity of FS-cells. In our next study, we found that FS-cells have the potential to differentiate into striated muscle cells. From FS-cells show multi-potent cell character and other cytological evidence, we propose that FS-cells are candidate of organ-specific stem cells in the anterior pituitary gland.     

Melanoma tumors frequently acquire LRP2/megalin expression,which modulates melanoma cell proliferation and survival rates

We show that the multiligand receptor megalin, known to mediate uptake and trafficking of nutrients and signaling molecules, is frequently expressed in malignant melanoma samples. Expression of megalin‐encoding mRNA was investigated in 65 samples of nevi, melanomas, and melanoma metastases and was observed in more than 60% of the malignant samples, while only in 20% of the benign counterparts. Megalin expression in nevus and melanoma samples was additionally investigated by immunohistochemistry, which confirmed our mRNA‐based observations. We furthermore show that a panel of tumor‐derived melanoma cell lines express LRP2/megalin endogenously. In these cells, megalin is internalized from the cell surface and localizes extensively to intracellular vesicles, confirming receptor activity and pointing toward association with the endocytic apparatus. Groundbreaking, our results indicate that sustained megalin expression in melanoma cells is crucial for cell maintenance, as siRNA‐mediated reduction in melanoma cell expression of LRP2/megalin significantly decreases melanoma cell proliferation and survival rates.     

Clusterin protects granulosa cells from apoptotic cell death during follicular atresia

Clusterin expression is associated with programmed cell death (apoptosis) in many cell types but its exact role has not yet been defined. This study was carried out to determine the cellular localization of clusterin in the ovary and its functional role in the apoptotic cell death of ovarian follicles. A homogenous population of healthy and atretic follicles was obtained by treating immature rats with pregnant mare serum gonadotropin (PMSG). Apoptotic cell death was evaluated by TUNEL. Clusterin expression in the healthy and atretic follicles was examined by immunohistochemical and Western blot analyses, and gene expression was examined by Northern blot analysis. Clusterin protein and its mRNA are only expressed in granulosa cells of atretic follicles obtained from PMSG-treated rats on day 5 of the treatment. Healthy follicles from PMSG-treated rats on day 2 of the treatment do not express clusterin. Theca and stroma cells of both healthy and atretic follicles showed no signs of apoptosis and did not express clusterin. Withdrawal of trophic support from granulosa cells in cultures to induce apoptosis resulted in a dramatic increase in the levels of clusterin and its mRNA compared to cells cultured in serum-supplemented medium. In an attempt to establish the functional role of clusterin in the apoptotic cell death of ovarian follicles, the biosynthesis of clusterin in granulosa cells of healthy follicles was blocked by treatment of cells with antisense oligonucleotide to its cDNA. Treatment of granulosa cells with the antisense oligonucleotide resulted in an increase in the apoptotic cell death compared to the control. These findings indicate that depletion of clusterin can lead to the programmed cell death in ovary, suggesting a functional role for this protein in follicular atresia.     

Soluble megalin is accumulated in the lumen of the rat endolymphatic sac

The endolymphatic sac (ES) is believed to play an important role in maintaining homeostasis in the inner ear by the absorption and endocytosis of endolymph. Megalin is a 600-kDa multiligand endocytic receptor expressed in certain types of absorptive epithelia including kidney proximal tubules. We analyzed the immunoreactivity for megalin in rat ES by immunofluorescence, immunogold electron microscopy, and immunoblotting. With immunostaining, the luminal substances of the ES were strongly stained for megalin. Megalin was also localized in luminal macrophage-like cells and both types of epithelial cell (mitochondria-rich cells and ribosome-rich cells). In these cells, the megalin was localized in the lumen of endosomes, but was not membrane associated. This localization pattern indicates that the megalin in these cells is not a membrane receptor, but merely one of the constituents that are endocytosed from the lumen of the ES. Immunoblotting indicated that the megalin in the ES is a 210-kDa molecule lacking a cytoplasmic domain. This suggests that the megalin in the ES may be a soluble form, different from the 600-kDa membrane-bound receptor expressed in kidneys. Taken together, it is likely that the megalin in the ES lumen is a soluble component and may be endocytosed by the ES epithelial cells. Furthermore, we found that the tectorial membrane, an acellular structure in the cochlea, gave a strong megalin immunoreaction. Since the cochlea is connected to the ES, the megalin may be transported alone or with the components of the tectorial membrane from the cochlea to the ES lumen through longitudinal flow.     

Immunohistochemical localization of huntingtin-associated protein 1 in endocrine system of the rat.

Huntingtin-associated protein 1 (HAP1) was originally found to be localized in neurons and is thought to play an important role in neuronal vesicular trafficking and/or organelle transport. Based on functional similarity between neuron and endocrine cell in vesicular trafficking, we examined the expression and localization of HAP1 in the rat endocrine system using immunohistochemistry. HAP1-immunoreactive cells are widely distributed in the anterior lobe of the pituitary, scattered in the wall of the thyroid follicles, or clustered in the interfollicular space of the thyroid gland, exclusively but diffusely distributed in the medullae of adrenal glands, and selectively located in the pancreas islets. HAP1-containing cells were also found in the mucosa of stomach and small intestine with a distributive pattern similar to that of gastrointestinal endocrine cells. However, no HAP1-immunoreactive cell was found in the cortex of the adrenal gland, the testis, and the ovary. In the posterior lobe of the pituitary, HAP1-immunoreactive products were not detected in the cell bodies but in many stigmoid bodies, one kind of non-membrane-bound cytoplasmic organelle with a central or eccentric electron-lucent core. HAP1-immunoreactive stigmoid bodies were also found in the cytoplasm of endocrine cells in the thyroid gland, the medullae of adrenal gland, the pancreas islets, the stomach, and small intestine. The present study demonstrates that HAP1 is selectively expressed in part of the small peptide-, protein-, and amino-acid analog and derivative-secreting endocrine cells but not in steroid hormone-secreting cells, suggesting that HAP1 is also involved in intracellular trafficking in certain types of endocrine cells.     

Expression profiles of apoptosis genes in mammary epithelial cells

To investigate apoptosis in HC11 mammary epithelial cells, we compared the gene expression profiles of actively growing and serum-starved apoptotic cells using a mouse apoptosis gene array and 33P-labeled cDNA prepared from the RNA of the two cultures. Analysis of the arrays showed that expression of several genes such as clusterin, secreted frizzled related protein mRNA (sFRP-1), CREB-binding protein (CBP), and others was higher in the apoptotic cells whereas expression of certain genes including survivin, cell division cycle 2 homolog A (CDC2), and cyclin A was lower. These expression patterns were confirmed by RT-PCR and/or Northern analyses. We compared the expression of some of these genes in the mouse mammary gland under various physiological conditions. The expression levels of genes (clusterin, CBP, and M6P-R) up-regulated in apoptotic conditions were higher at involution than during lactation. On the other hand, genes (Pin, CDC2) downregulated in apoptotic conditions were relatively highly expressed in virgin and pregnant mice. We conclude that certain genes such as clusterin, sFRP-1, GAS1 and CBP are induced in apoptotic mammary epithelial cells, and others are repressed. Moreover, the apoptosis array is an efficient technique for comparing gene expression profiles in different states of the same cell type.     

Megalin and cubilin expression in gallbladder epithelium and regulation by bile acids

Cholesterol crystal formation in the gallbladder is a key step in gallstone pathogenesis. Gallbladder epithelial cells might prevent luminal gallstone formation through a poorly understood cholesterol absorption process. Genetic studies in mice have highlighted potential gallstone susceptibility alleles, Lith genes, which include the gene for megalin. Megalin, in conjunction with the large peripheral membrane protein cubilin, mediates the endocytosis of numerous ligands, including HDL/apolipoprotein A-I (apoA-I). Although the bile contains apoA-I and several cholesterol-binding megalin ligands, the expression of megalin and cubilin in the gallbladder has not been investigated. Here, we show that both proteins are expressed by human and mouse gallbladder epithelia. In vitro studies using a megalin-expressing cell line showed that lithocholic acid strongly inhibits and cholic and chenodeoxycholic acids increase megalin expression. The effects of bile acids (BAs) were also demonstrated in vivo, analyzing gallbladder levels of megalin and cubilin from mice fed with different BAs. The BA effects could be mediated by the farnesoid X receptor, expressed in the gallbladder. Megalin protein was also strongly increased after feeding a lithogenic diet. These results indicate a physiological role for megalin and cubilin in the gallbladder and provide support for a role for megalin in gallstone pathogenesis.     

Antisense Oligonucleotide of Clusterin mRNA Induces Apoptotic Cell Death and Prevents Adhesion of Rat ASC-17D Sertoli Cells

Clusterin has been known to play important roles in cell-cell and/or cell-substratum interactions. Recently we reported the transient expression of clusterin in pancreatic endocrine cells during the early developmental stages and suggested a role in aggregating the endocrine cells for islet formation. In the present study, we have investigated the involvement of clusterin in cell-substratum interaction by the inhibition of clusterin synthesis using antisense oligonucleotide. The expression of clusterin was transiently increased as early as 2–8 h after plating the ASC-17D Sertoli cells to the culture flask, which was the period of cell attachment. In addition, up-regulation of clusterin mRNA was so much greater when the Sertoli cells were plated on the petri dish for the bacterial culture instead of in a animal cell culture flask that therefore, the cells failed to attach to it. These findings suggested that interruption of cell to plate substratum interaction might lead to over-expression of clusterin from Sertoli cells to induce cell to cell aggregation or, perhaps, to re-establish attachment with the substratum. Transfection of ASC-17D Sertoli cells with a 20-base antisense oligonucleotide against clusterin mRNA resulted in extracellular release of LDH and DNA fragmentation. Sertoli cell death by antisense oligonucleotide of clusterin was sequence specific and dose dependent. Treatment of antisense oligonucleotide induced a marked reduction of synthesis for clusterin protein, but not for clusterin mRNA expression, suggesting the translational suppression of clusterin by antisense oligonucleotide. Further, microscopic observation showed that more noticeable cell death was induced by treating the antisense prior to plating the cells than by treating after cell attachment to the plate. From these results, we speculate that down-regulation of clusterin expression in the anchorage-dependent Sertoli cells prevents them from attaching to the plate, and therefore induces cell death.     

Clusterin activates survival through the phosphatidylinositol 3-kinase/Akt pathway

Clusterin is, in its major form, a secreted heterodimeric disulfide-linked glycoprotein (75-80 kDa). It was first linked to cell death in the rat ventral prostate after androgen deprivation. Recent studies have demonstrated that overexpression of clusterin in prostatic cells protects them against tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. However the details of this survival mechanism remain undefined. Here, we investigate how clusterin prevents cells from undergoing TNFalpha-induced apoptosis. We established a double-stable prostatic cell line for inducible clusterin by using the Tet-On gene expression system. We demonstrated that 50% of the cells overexpressing clusterin escaped from TNFalpha- and actinomycin D-induced cell death. Moreover we demonstrated that the incubation of MLL cells with conditioned medium containing the secreted clusterin or the supplementation of purified clusterin in the extracellular medium decreased the TNFalpha-induced apoptosis significantly. This extracellular action implicates megalin, the putative membrane receptor for clusterin to mediate survival. Indeed clusterin overexpression up-regulated the expression of megalin and induced its phosphorylation in a dose-dependent manner. We interestingly showed that clusterin overexpression is associated with the up-regulation of the phosphorylation of Akt. Activated Akt induced the phosphorylation of Bad and caused a decrease of cytochrome c release. These results enable us to pinpoint one mechanism by which secreted clusterin favors survival in androgen-independent prostate cancer cells, implicating its receptor megalin and Akt survival pathway.     

Neuropeptide W is expressed in the noradrenalin-containing cells in the rat adrenal medulla

Neuropeptide W (NPW) is an endogenous ligand for GPR7, a member of the G-protein-coupled receptor family. NPW plays an important role in the regulation of both feeding and energy metabolism, and is also implicated in modulating responses to an acute inflammatory pain through activation of the hypothalamus-pituitary-adrenal axis. GPR7 mRNA has been shown to be expressed in the hypothalamus, pituitary gland and adrenal cortex. Similarly, NPW expression has been demonstrated in the brain and pituitary gland. However, the precise distribution of NPW-producing cells in the adrenal gland remains unknown. The aim of this study was to explore the distribution and localization of NPW immunoreactivity in the rat adrenal gland. Total RNA was prepared from the hypothalamus, pituitary gland and adrenal gland. RT-PCR revealed the expression of NPW mRNA in these tissues, while in situ hybridization demonstrated the presence of NPW mRNA in the adrenal medulla. When immunohistochemistry was performed on sections of adrenal gland, NPW-like immunoreactivity (NPW-LI) was observed in the medulla but not in the cortex. Moreover, NPW-LI was found to be co-localized in cells which expressed dopamine beta hydroxylase but not phenylethanolamine-N-methyltransferase. The finding that NPW is expressed in noradrenalin-containing cells in the adrenal medulla suggests that it may play an important role in endocrine function in the adrenal gland.     

On the expression of cytokeratins and their distribution in some rabbit gland tissues.

The composition and distribution of cytokeratins were studied in a series of exocrine and endocrine glands in the rabbit, by means of anti-human monoclonal antibodies. Rabbit cytokeratins are known to be homologous to those in man, and their in situ distribution was also found to be similar. In acinic cells of the exocrine glands, only cytokeratins 8 and 18 were detected, whereas in ductal cells 1, 5, 10, 11 and 13, 19 were also present. The myoepithelial cells surrounding acini and basal duct cells were stained with the antibodies directed against cytokeratins 13 and 1, 5, 10, 11. Among the endocrine glands, a higher reactivity was observed in the thyroid gland and pancreatic islets, while pituitary and adrenal gland often remained unstained. Although the expression of cytokeratins was found to be almost homogeneous in each glandular cell type, a certain variability was observed, depending on the organ examined and the treatment carried out.     

Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.     

The antisecretory factor: synthesis and intracellular localisation in porcine tissues

The antisecretory factor, AF, is a 41-kDa protein, cloned and sequenced from a human pituitary library. AF is a potent inhibitor of experimental intestinal hypersecretion in rats and pigs. An antiserum against the C-terminal of the truncated, recombinantly produced AF protein was raised in rabbits. The affinity-purified antiserum was used to study the expression of AF in mucosal membranes and in the pituitary gland of the pig; distinctly stained cells were found in lymphoid cells in the connective tissue of all parts of the gastrointestinal, respiratory and urinary tracts. Cytoplasmic AF was demonstrated in endocrine and epithelial cells in the pituitary gland. In situ hybridisation with a digoxigenin-labelled mRNA probe also demonstrated specific cytoplasmic staining in epithelial and lymphoid cells in all of these tissues. The cells stained by either method were similarly distributed topographically within the tissues. The results suggest that a specific defined cell population in these various tissues possesses the capability of both synthesising and storing the AF protein within the cellular cytoplasmic compartment.     

激活素受体相互作用蛋白1在小鼠组织中的表达与分布

激活素具有调节激素分泌以及神经保护等多种作用,最近在小鼠脑内发现的激活素受体相互作用蛋白1(ARIP1)具有介导激活素信号传导作用,但有关ARIP1的分布情况仍然不清楚。本研究采用RT-PCR及免疫组织化学染色分析ARIP1在脑及脑外的表达与分布情况。RT-PCR检测发现ARIP1 mRNA不仅在大脑、小脑表达,在垂体、肾上腺以及睾丸也有明显表达。免疫组化染色显示大脑、小脑、垂体、肾上腺和睾丸均有不同程度的ARIP1免疫染色反应,小脑中浦肯野细胞着色明显,大脑主要是海马和下丘脑,在神经垂体、腺垂体的嗜碱细胞以及肾上腺网状带、球状带、束状带中均有表达,睾丸间质细胞也可见ARIP1成熟蛋白表达。结果提示,ARIP1不仅参与脑神经细胞的信号传导调节,也可能参与神经内分泌腺的功能调节。     

Expression of retinaldehyde dehydrogenase 1 in the anterior pituitary glands of adult rats

Retinoic acid (RA) plays a critical role in cell growth and tissue development and is also a regulatory factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine factor is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases that catalyze the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. Recently, we demonstrated that RALDH2 and RALDH3, but not RALDH1, were expressed in the developing anterior pituitary gland of rats, but the expression of RALDHs in the adult pituitary gland was not determined. Therefore, we have now examined the expression of RALDH1, RALDH2, and RALDH3 mRNAs in the pituitary gland of adult rats. Analysis by quantitative real-time polymerase chain reaction of adult pituitary glands has revealed a high level of RALDH1 mRNA but not of RALDH2 mRNA or RALDH3 mRNA. We have also detected mRNA expression for RALDH1 in the anterior pituitary gland by in situ hybridization with digoxigenin-labeled cRNA probes. Double-staining for RALDH1 mRNA and pituitary hormones or S-100 protein, a marker of folliculo-stellate cells (FS-cells), has revealed RALDH1 mRNA expression in a portion of prolactin-producing cells, marginal layer cells, and FS-cells. Our results suggest that RA is generated in the adult anterior pituitary gland, and that it may act locally on pituitary cells. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (18790149) and by the Foundation of Growth Science.     

The adaptor protein ARH escorts megalin to and through endosomes

Megalin is an endocytic receptor that binds multiple ligands and is essential for many physiological processes such as brain development and uptake of proteins by the kidney tubule, yolk sac, and thyroid. The cytoplasmic tail of megalin contains two FXNPXY motifs. Autosomal recessive hypercholesterolemia (ARH) is an adaptor protein that binds to the FXNPXY motif of the low-density lipoprotein receptor as well as clathrin and AP-2. We found that ARH also binds to the first FXNPXY motif of megalin in two-hybrid, pull-down and coimmunoprecipitation assays. ARH colocalizes with megalin in clathrin coated pits and in recycling endosomes in the Golgi region. When cells are treated with nocodazole, the recycling endosomes containing megalin and ARH disperse. On internalization of megalin, ARH and megalin are first seen in clathrin coated pits followed by sequential localization in early endosomes and tubular recycling endosomes in the pericentriolar region followed by their reappearance at the cell surface. Expression of ARH in Madin-Darby canine kidney cells expressing megalin mini-receptors enhances megalin-mediated uptake of 125I-lactoferrin, a megalin ligand. These results show that ARH facilitates endocytosis of megalin, escorts megalin along its endocytic route and raise the possibility that transport through the endosomal system is selective and requires interaction with specific adaptor proteins.