LRH-1在肿瘤及干细胞中的功能研究进展

核受体(nuclear receptors,NRs)是转录因子家族中最大的成员,多以配体依赖的方式特异性调节其靶基因的表达,参与机体代谢、发育和生殖功能的调控。LRH-1(liver receptor homolog-1),也称为NR5A2(nuclear receptor subfamily 5,group A,member 2),是核受体家族的成员,作为转录共激活子调控相关基因的表达。LRH-1调控多种重要的生理功能,包括调节脂肪酸和胆固醇的代谢,另外在胚胎发育和分化中也起了重要作用。LRH-1在促进多种癌症的发生过程中扮演重要的角色,如结肠癌、胰腺癌、卵巢癌和乳腺癌。随着对LRH-1研究的深入,其在疾病和胚胎干细胞中的功能作用已备受关注,这也使得LRH-1成为了许多疾病的潜在治疗靶点。     

Immunolocalization of aquaporin 1, 5, and 9 in the female pig reproductive system

Thirteen mammalian aquaporin (AQP) isoforms have been identified, and they have a unique tissue-specific pattern of expression. AQPs have been documented in the reproductive system of both male and female humans, rats, and mice. However, tissue expression and cellular and subcellular localization of AQPs are unknown in the female reproductive system of pigs. In this study, AQP1 immunoreactivity was detected in the capillary endothelium of the ovary. Distinct immunolabeling of capillary endothelium was also observed in the oviduct and uterus. AQP5 was expressed in flattened follicle cells of primordial follicles, granulosa cells of developing ovarian follicles, and muscle cells of the oviduct and uterus. Staining of AQP5 was also observed in the epithelial cells of the oviduct and uterine epithelium. AQP9 immunoreactivity was observed in granulosa cells of developing follicles. AQP9 was also localized in the luminal epithelial cells of the oviduct and uterine epithelia cells. This is, to our knowledge, the first study that shows tissue expression and cellular and subcellular localization of AQPs in the reproductive system of the female pig. Moreover, these results suggest that several subtypes of the AQPs (AQP1, 5, and 9) are involved in regulation of water homeostasis in the reproductive system of gilts.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.     

Phosphorylation of the hinge domain of the nuclear hormone receptor LRH-1 stimulates transactivation

The nuclear receptor LRH-1 (NR5A2) functions to regulate expression of a number of genes associated with bile acid homeostasis and other liver functions, but mechanisms that modulate its activity remain unclear. We have found that mitogenic stimuli, including treatment with phorbol myristate (PMA), increase LRH-1 transactivation. This response maps to the hinge and ligand binding domains of LRH-1 and is blocked by the mitogen-activated protein kinase ERK1/2 inhibitor U0126. LRH-1 is a phosphoprotein and hinge domain serine residues at 238 and 243 are required for effective phosphorylation, both in vitro and in cells. Preventing phosphorylation of these residues by mutating both to alanine decreases PMA-dependent LRH-1 transactivation and mimicking phosphorylation by mutation to positively charged aspartate residues increases basal transactivation. Although serine phosphorylation of the hinge of SF-1 (NR5A1), the closest relative of LRH-1, confers a similar response, the specific targets differ in the two closely related orphan receptors. These results define a novel pathway for the modulation of LRH-1 transactivation and identify specific LRH-1 residues as downstream targets of mitogenic stimuli. This pathway may contribute to recently described proliferative functions of LRH-1.     

LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner

Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53.     

Effects of bone morphogenetic proteins on epithelial repair

Epithelial tissue has important functions such as protection, secretion, and sensation. Epithelial damage is involved in various pathological processes. Bone morphogenetic proteins (BMPs) are a class of growth factors with multiple functions. They play important roles in epithelial cells, including in differentiation, proliferation, and migration during the repair of the epithelium. This article reviews the functions and mechanisms of the most profoundly studied BMPs in the process of epithelial damage repair and their clinical significance.     

Apical accumulation of the Drosophila PDGF/VEGF receptor ligands provides a mechanism for triggering localized actin polymerization

Epithelial tissue functions depend largely on a polarized organization of the individual cells. We examined the roles of the Drosophila PDGF/VEGF receptor (PVR) in polarized epithelial cells, with specific emphasis on the wing disc epithelium. Although the receptor is broadly distributed in this tissue, two of its ligands, PVF1 and PVF3 are specifically deposited within the apical extracellular space, implying that polarized apical activation of the receptor takes place. The apical localization of the ligands involves a specialized secretion pathway. Clones for null alleles of Pvr or expression of RNAi constructs showed no phenotypes in the wing disc or pupal wing, suggesting that Pvr plays a redundant role in this tissue. However, when uniform expression of a constitutively dimerizing receptor was induced, loss of epithelial polarity, formation of multiple adherens and septate junctions, and tumorous growth were observed in the wing disc. Elevation of the level of full-length PVR also gave rise to prominent phenotypes, characterized by higher levels of actin microfilaments at the basolateral areas of the cells and irregular folding of the tissue. Together, these results suggest that polarized PVR activation is necessary for the proper organization of the wing disc epithelium, by regulating the apical assembly of the actin cytoskeleton.     

The synthesis and localization of crystallins in different cell compartments of the crystalline lens in adult frogs: immunoautoradiographic and immunofluorescent research

The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (Mikha?lov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.     

Perilipin 5 is a novel target of nuclear receptor LRH-1 to regulate hepatic triglycerides metabolism

Liver receptor homolog-1 (LRH-1) has emerged as a regulator of hepatic glucose, bile acid, and mitochondrial metabolism. However, the functional mechanism underlying the effect of LRH-1 on lipid mobilization has not been addressed. This study investigated the regulatory function of LRH-1 in lipid metabolism in maintaining a normal liver physiological state during fasting. The Lrh-1^f/f and LRH-1 liver-specific knockout (Lrh-1^LKO) mice were either fed or fasted for 24 h, and the liver and serum were isolated. The livers were used for qPCR, western blot, and histological analysis. Primary hepatocytes were isolated for immunocytochemistry assessments of lipids. During fasting, the Lrh-1^LKO mice showed increased accumulation of triglycerides in the liver compared to that in Lrh-1^f/f mice. Interestingly, in the Lrh-1^LKO liver, decreases in perilipin 5 (PLIN5) expression and genes involved in β-oxidation were observed. In addition, the LRH-1 agonist dialauroylphosphati-dylcholine also enhanced PLIN5 expression in human cultured HepG2 cells. To identify new target genes of LRH-1, these findings directed us to analyze the Plin5 promoter sequence, which revealed −1620/−1614 to be a putative binding site for LRH-1. This was confirmed by promoter activity and chromatin immuno-precipitation assays. Additionally, fasted Lrh-1^f/f primary hepatocytes showed increased co-localization of PLIN5 in lipid droplets (LDs) compared to that in fasted Lrh-1^LKO primary hepatocytes. Overall, these findings suggest that PLIN5 might be a novel target of LRH-1 to mobilize LDs, protect the liver from lipid overload, and manage the cellular needs during fasting.     

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.     

Liver receptor homolog 1 is a negative regulator of the hepatic acute-phase response

The orphan nuclear receptor liver receptor homolog 1 (LRH-1) has been reported to play an important role in bile acid biosynthesis and reverse cholesterol transport. Here, we show that LRH-1 is a key player in the control of the hepatic acute-phase response. Ectopic expression of LRH-1 with adenovirus resulted in strong inhibition of both interleukin-6 (IL-6)- and IL-1beta-stimulated haptoglobin, serum amyloid A, and fibrinogen beta gene expression in hepatocytes. Furthermore, induction of the hepatic inflammatory response was significantly exacerbated in HepG2 cells expressing short hairpin RNA targeting LRH-1 expression. Moreover, transient-transfection experiments and electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that LRH-1 regulates this cytokine-elicited inflammatory response by, at least in part, antagonizing the CCAAT/enhancer binding protein beta signaling pathway. Finally, we show, by using LRH-1 heterozygous mice, that LRH-1 is involved in the control of the inflammatory response at the hepatic level in vivo. Taken together, our results outline an unexpected role for LRH-1 in the modulation of the hepatic acute-phase response.