Calcium phosphate and calcium oxalate crystal handling is dependent upon CLC-5 expression in mouse collecting duct cells

Defects in an intracellular chloride channel CLC-5 cause Dent's disease, an inherited kidney stone disorder. Using a collecting duct model, mIMCD-3 cells, we show expression of dimeric mCLC-5. Transient transfection of antisense CLC-5 reduces CLC-5 protein expression. Binding of both calcium phosphate (hydroxyapatite) and calcium oxalate monohydrate (COM) crystals overlaid onto mIMCD-3 cultures was affected by altered CLC-5 expression. Calcium phosphate crystal agglomerations (>10 microm) were minimal in control (9%) and sense (13%) CLC-5-transfected cells, compared to 66% of antisense CLC-5-transfected cells (P<0.001). Small calcium phosphate crystals (<10 microm) were found associated with 45% of sense CLC-5-treated cells, of which the majority (11/14 cells) appeared to be internalised within the cell. Calcium oxalate agglomerations (>10 microm) were also largely absent for controls or sense mCLC-5 transfectants (11% and 9% of cells, respectively) with COM crystal agglomerates predominating in antisense CLC-5 transfectants (66%, P<0.0001). We conclude that collecting duct cells with reduced CLC-5 expression lead to a tendency to form calcium crystal agglomeration, which may help explain the nephrocalcinosis and nephrolithiasis seen in Dent's disease.     

A role for CBS domain 2 in trafficking of chloride channel CLC-5

CLC-5 is a member of the CLC family of voltage-gated chloride channels. Mutations disrupting CLC-5 lead to Dent's disease, an X-linked renal tubular disorder, characterised by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal stones. Sequence analysis of CLC-5 reveals a 746 amino acid protein with an intracellular amino-terminus, transmembrane spanning domains, and two CBS domains within its intracellular carboxy-terminus. CBS domains have been implicated in intracellular targetting and trafficking as well as protein-protein interactions. We investigate subcellular localisation of three naturally occurring CLC-5 mutants which all lead to a truncated protein, disrupting the second CBS domain. These mutants are unable to traffic normally to acidic endosomes but are retained in perinuclear compartments, colocalising with the Golgi complex. This is the first identification of the cellular pathogenesis of CBS domain mutations of CLC-5.     

Effects of basic fibroblast growth factor on angiogenin expression and cell proliferation in H7402 human hepatoma cells

Hepatocellular carcinoma （HCC） is one of the most common cancers worldwide. Basic fibroblast growth factor （bFGF）, which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Its expression is essential for the progression and metastasis of HCC. This study aims to investigate the effects of bFGF on the expression of angiogenin, another growth factor, which plays an important role in tumor angiogenesis, and on cell proliferation in H7402 human hepatoma cells. The bFGF sense cDNA or antisense cDNA was stably transfected into H7402 cells. Genomic DNA PCR analysis demonstrated that human bFGF sense cDNA or antisense cDNA was inserted into the genome. Furthermore, the expression of bFGF and angiogenin was examined by RT-PCR and Western blot assays. MTT and colony formation assays were employed to determine cell proliferation. Stable bFGF over-expressing and under-expressing transfectants were successfully established. Expression of angiogenin was decreased in the over-expressing bFGF cells （sense transfectants） and was increased in the under-expressing bFGF cells （antisense transfectants）. Cell proliferation increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results demonstrated that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. This study may be helpful in finding a potential therapeutic approach to HCC.     

Up-regulation of adipogenesis in adipocytes expressing stably cyclooxygenase-2 in the antisense direction

Adipocytes and the precursor cells express two types of cyclooxygenase (COX) isoforms that are involved in the biosynthesis of different types of prostaglandins (PGs) exerting opposite effects on adipogenesis. To evaluate the role of the inducible COX-2 isoform in the control of the differentiation and maturation of adipocytes, we employed an antisense technology to suppress specifically the expression of COX-2 in adipocytes. Cultured 3T3-L1 preadipocytes were transfected stably with a mammalian expression vector having the full-length cDNA encoding mouse COX-2 oriented in the antisense direction. The cloned transfectants with antisense COX-2 exhibited stable expression of antisense RNA for COX-2, which was accompanied by the suppressed expression of mRNA and protein levels of sense COX-2. However, almost no alteration in the expression of COX-1 was detected. The transfectants with antisense COX-2 showed significant decreases in the delayed synthesis of PGE₂ involving the inducible COX-2 in response to cell stimuli. By contrast, the immediate synthesis of PGE₂ associated with the constitutive COX-1 was not influenced appreciably. The stable expression of antisense mRNA of COX-2 resulted in significant stimulation of fat storage during the maturation phase without affecting the cell proliferation associated with the clonal expansion phase. The gene expression studies revealed higher expression levels of adipocyte-specific markers in the transfectants with antisense COX-2, indicating the mechanism that stimulates adipogenesis program. The up-regulation of fat storage was appreciably prevented by anti-adipogenic prostanoids, such as PGE₂ and PGF_2α, during the maturation phase. These results suggest that COX-2 is more preferentially involved in the generation of endogenous anti-adipogenic prostanoids during the maturation phase of adipocytes.     

Sense and antisense modification of glial alpha B-crystallin production results in alterations of stress fiber formation and thermoresistance

The phenotypic effects of selectively altering the levels of alpha B- crystallin in cultured glial cells were analyzed using sense and antisense approaches. Rat C6 glioma cells and human U-373MG glioma cells were transfected with a rat alpha B-crystallin sense cDNA or an antisense cDNA regulated by a Rous sarcoma virus promoter to alter cellular levels of alpha B-crystallin. The antisense strategy resulted in decreased alpha B-crystallin levels, as revealed by Western blot and immunocytochemical analyses. The reduced alpha B-crystallin expression was accompanied by alterations in cellular phenotype: (a) a reduction of cell size and/or a slender cell morphology; (b) a disorganized microfilament network; and (c) a reduction of cell adhesiveness. Like HSP27, the presence of additional alpha B-crystallin protein confers a thermoresistant phenotype to stable transfectants. Thus, alpha B- crystallin in glioma cells plays a role in their thermal resistance and may contribute to the stability of cytoskeletal organization.     

Isolation and characterization of the human CLC-5 chloride channel gene promoter

The human CLC-5 chloride channel is expressed mainly in the kidney and its mutations cause Dent's disease (a familial renal tubular syndrome with hypercalciuria, tubular proteinuria, rickets, nephrocalcinosis, and eventual renal failure). To gain insight into the regulatory mechanism of CLC-5 expression, a genomic clone that contains the 5'-flanking region of the human CLC-5 gene was isolated and characterized. Two types of 5'-ends of cDNA were isolated by 5'-rapid amplification of cDNA ends, and one of them, approximately 2.1 kbp upstream of ATG-containing exon II, was first identified in human. The major promoter activity was detected in the 5'-flanking region of this newly identified exon Ia. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-1-like site and cAMP-responsive element, but it lacked a TATA box, a GC-rich element, and an SP-1 site. Deletion analysis of the 5'-flanking region showed that the fragments containing the AP-1-like element (TGACTCC) positioned at -38 exhibited high promoter activities in CLC-5 expressing LLC-PK1 cells, but that further deletions not containing this AP-1-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-1-like element in LLC-PK1 cells, which seemed to differ from an authentic AP-1. This study clarified the key element of the human CLCN5 promoter, and the mutation in this region could be the cause of Dent's disease.     

Stable transfection of DAOY cells with a GM3 synthase antisense construct and transient reduction in ganglioside content

Tumor cell gangliosides are bioactive molecules involved in tumor-host interactions. To investigate their role in tumor formation and angiogenesis, we sought to develop an inhibitory model targeting human GM3 synthase, an essential enzyme in the ganglioside synthesis pathway, by antisense transfection. We prepared a number of transfectants from DAOY human medulloblastoma cells and isolated clones that stably expressed a 560-bp fragment of human GM3 synthase cDNA, in either sense or antisense orientation, as well as clones transfected with an empty vector. Both sense and antisense clones permanently incorporated mammalian expression vectors into their genomes. The DAOY cell clones were screened for ganglioside content using total lipid extraction, ganglioside isolation, and HPTLC. One antisense-transfected clone, 7.2A, in which total ganglioside content was reduced by 70%, was selected for further study. All sense-and sham-transfectants had ganglioside levels not different from that of untransfected DAOY cells. After 10 passages however, while antisense mRNA expression was fully maintained, the ganglioside content of 7.2A cells had reverted to normal levels. Antisense RNA transfection can sometimes have a reversible effect on the expression of a target. Possible regulatory mechanisms of this previously unrecognized process of reversion to wild type phenotype are discussed.     

Antisense CD11b integrin inhibits the development of a differentiated monocyte/macrophage phenotype in human leukemia cells

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.     

Mxi1 influences cyst formation in three-dimensional cell culture

Cyst formation is a major characteristic of ADPKD and is caused by the abnormal proliferation of epithelial cells. Renal cyst formation disrupts renal function and induces diverse complications. The mechanism of cyst formation is unclear. mIMCD-3 cells were established to develop simple epithelial cell cysts in 3-D culture. We confirmed previously that Mxi1 plays a role in cyst formation in Mxi1-deficient mice. Cysts in Mxi1 transfectanted cells were showed by collagen or mebiol gels in 3-D cell culture system. Causative genes of ADPKD were measured by q RT-PCR. Herein, Mxi1 transfectants rarely formed a simple epithelial cyst and induced cell death. Overexpression of Mxi1 resulted in a decrease in the PKD1, PKD2 and c-myc mRNA relating to the pathway of cyst formation. These data indicate that Mxi1 influences cyst formation of mIMCD-3 cells in 3-D culture and that Mxi1 may control the mechanism of renal cyst formation. [BMB reports 2012; 45(3): 189-193].     

Antisense GAP-43 Inhibits the Evoked Release of Dopamine from PC12 Cells

Abstract: To investigate the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, F1, P-57) in neurotransmitter release, we transfected PC12 cells with a recombinant expression vector coding for antisense human GAP-43 cRNA. Two stable transfectants, designated AS1 and AS2, were selected that had integrated the recombinant sequence and expressed antisense GAP-43 RNA. Immunoblot analysis of proteins from AS1 and AS2 cells indicated that the level of GAP-43 in these cell lines was reduced. In the presence of extracellular calcium, a depolarizing concentration of K⁺ (56 m M ) evoked dopamine release from control cells, but not from AS1 and AS2 cells. Similarly, the calcium ionophore A23187 evoked dopamine release from control cells, but was ineffective in stimulating dopamine release from AS1 and AS2 cells. The antisense transfectants, as well as the control cells, contained appreciable quantities of dopamine and secretory granules with a normal appearance. Because the expression of antisense GAP-43 RNA in PC12 cells leads to a decrease in GAP-43 expression and to the loss of evoked dopamine release, these results provide evidence of a role for GAP-43 in calcium-dependent neurotransmitter release.     

Inhibition of glucocorticoid-induced apoptosis by the expression of antisense gene of mitochondrial ATPase subunit 6(1)

To isolate the apoptosis-linked genes involved in the cell death of thymocytes induced by glucocorticoids, we developed a functional cloning assay. Murine CD4(+)CD8(+) thymic cell line 2-257-20 cells were transfected with cDNA expression libraries obtained from a dexamethasone-resistant cell line. The transfected cells were selected in the presence of dexamethasone, and the plasmids which episomally expanded were then extracted from the surviving cells. One of the rescued cDNAs was found to be an antisense cDNA fragment identical to the mouse mitochondrial ATPase 6 gene. In the stable transfectants with the ATPase 6 antisense gene, the induction of apoptosis by dexamethasone was significantly delayed. Furthermore, the ATP synthesis in these transfectants was also reduced to some extent. ATPase 6 is a subunit of F(o)F(1) ATPase and our results support that ATP synthesis from the mitochondria is necessary for the induction of apoptosis induced by glucocorticoids.     

NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype.

In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.     

Transformation of NIH 3T3 cells by enhanced PAR expression

Prostate androgen regulated (PAR) is a 1038bp novel gene located on chromosome 1 in epidermal differentiation complex. The gene is ubiquitously expressed in normal tissues and is overexpressed in most of their malignant counterparts. PAR cellular function is unknown. Here we report the effect of increased PAR expression induced by transfection of PAR cDNA on NIH3T3 cell phenotype. PAR-NIH3T3 transfectants expressing 3- to 4-fold higher PAR levels compared to controls grew faster in tissue cultures, formed colonies in soft agar, and exhibited a shortening of G1 and S phases of cell cycle and formed tumors in SCID mice. Transfection of NIH3T3 cells with increased ectopic PAR expression with a 22 mer oligonucleotide in antisense orientation with PAR mRNA abrogated their ability to form colonies in soft agar. The data presented here along with our previously reported results on DU145 cells transfected with antisense PAR cDNA suggest that PAR gene behaves like a proto-oncogene.     

Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts

Normal lung morphogenesis is dependent on chloride-driven fluid transport. The molecular identity of essential fetal lung chloride channel(s) has not been elucidated. CLC-2 is a chloride channel, which is expressed on the apical surface of the developing respiratory epithelium. CLC-2-like pH-dependent chloride secretion exists in fetal airway cells. We used a 14-day fetal rat lung submersion culture model to examine the role of CLC-2 in lung development. In this model, the excised fetal lung continues to grow, secrete fluid, and become progressively cystic in morphology (26). We inhibited CLC-2 expression in these explants, using antisense oligonucleotides, and found that lung cyst morphology was disrupted. In addition, transepithelial voltage (V(t)) of lung explants transfected with antisense CLC-2 was inhibited with V(t) = -1.5 +/- 0.2 mV (means + SE) compared with -3.7 +/- 0.3 mV (means + SE) for mock-transfected controls and -3.3 +/- 0.3 mV (means + SE) for nonsense oligodeoxynucleotide-transfected controls. This suggests that CLC-2 is important for fetal lung fluid production and that it may play a role in normal lung morphogenesis.