Identification of genes differentially expressed in benign prostatic hyperplasia.

Differences between benign prostatic hyperplasia (BPH) and normal prostate tissue at the level of mRNA expression provide an opportunity to identify candidate genes for this disease. A cDNA subtraction procedure was used to isolate differentially expressed genes in BPH. The subtraction was done by solution hybridization of BPH cDNA against excess normal prostate cDNA. We identified known, EST, and novel genes by sequence and database analysis of the subtracted cDNAs. Several of these cDNAs were used as probes in Northern blotting analysis to confirm over-expression of their corresponding mRNAs in BPH tissues. One highly upregulated sequence of interest shared identity with a known mRNA encoding human NELL2, a protein containing epidermal growth factor-like domains. NELL2 was not previously reported to be expressed in prostate and may code for a novel prostatic growth factor. In situ hybridization analysis of hyperplastic prostate specimens demonstrated that NELL2 mRNA expression is predominantly localized in basal cells of the epithelium. Disease-related changes in the levels of NELL2 may contribute to alterations in epithelial-stromal homeostasis in BPH. (J Histochem Cytochem 49:669-670, 2001)     

Nucleotide Sequence and Regulatory Studies of VGF, a Nervous System-Specific mRNA That Is Rapidly and Relatively Selectively Induced by Nerve Growth Factor

A nervous system-specific mRNA that is rapidly induced in PC12 cells to a greater extent by nerve growth factor (NGF) than by epidermal growth factor treatment has been cloned. The polypeptide deduced from the nucleic acid sequence of the NGF33.1 cDNA clone contains regions of amino acid sequence identity with that predicted by the cDNA clone VGF, and further analysis suggests that both NGF33.1 and VGF cDNA clones very likely correspond to the same mRNA (VGF). In this report both the nucleic acid sequence that corresponds to VGF mRNA and the polypeptide predicted by the NGF33.1 cDNA clone are presented. Genomic Southern analysis and database comparison did not detect additional sequences with high homology to the VGF gene. Induction of VGF mRNA by depolarization and phorbol 12-myristate 13-acetate treatment was greater than by serum stimulation or protein kinase A pathway activation. These studies suggest that VGF mRNA is induced to the greatest extent by NGF treatment and that VGF is one of the most rapidly regulated neuronal mRNAs identified in PC12 cells.     

Analysis of gene expression during differentiation of adipogenic cells in culture and hormonal control of the developmental program

Treatment of 10T1/2 mouse embryo fibroblasts with 5-azacytidine, an inhibitor of mammalian DNA methylation, leads to the appearance of several new cell types, including adipocytes. We have isolated several such adipogenic cell lines and characterized two of them, TA1 and TA2. When subconfluent these cells resemble fibroblasts. After growth is arrested at high density, both clones express a functional adipose phenotype characterized by accumulation of lipid droplets. This in vitro differentiation is accompanied by a greater than 100-fold increase in glycerol phosphate dehydrogenase activity, an enzyme characteristic of mature adipocytes. Consistent with these morphologic and enzymatic changes, differentiated TA1 cells show a widespread alteration in protein composition as well as a substantial change in the pattern of secreted proteins. We have constructed a cDNA library of TA1 adipocytes and have isolated 12 different cDNA clones corresponding to mRNAs that are induced during adipogenesis. Among these RNAs, some are not expressed prior to initiating differentiation whereas others are expressed in 10T1/2 cells and TA1 preadipocytes. Treatment of TA1 cells with insulin and the synthetic glucocorticoid dexamethasone leads to an acceleration of the phenotypic changes observed during adipogenesis. We have found that hormone treatment leads to a precocious accumulation of specific RNA for all of the clones studied. Analysis of the temporal control of RNA accumulation during differentiation indicates that there are different categories of RNAs, some of which accumulate by day 1 after treatment while others are not apparent until day 3.     

Expression and regulation of pOb24 and lipoprotein lipase genes during adipose conversion

Lipoprotein lipase (LPL) and pOb24 mRNAs are known to be early markers of adipose cell differentiation. Comparative studies of the expression of pOb24 and LPL genes during adipose conversion of Ob1771 preadipocyte cells and in mouse adipose tissue have shown the following: 1) the expression of both genes takes place at confluence; this event can also be triggered by growth arrest of exponentially growing cells at the G1/S stage of the cell cycle; 2) In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 and lipoprotein lipase mRNAs requires neither growth hormone or tri-iodothyronine as obligatory hormones nor insulin as a modulating hormone; 3) in mouse adipose tissue, pOb24 mRNA is present at a high level in stromal-vascular cells and at a low level in mature adipocytes, and in contrast LPL mRNAs are preferentially expressed in mature adipocytes. Thus, these two genes do not appear to be regulated in a similar manner, as also shown by the differential inhibition of their expression by tumor necrosis factor (TNF) and transforming growth factor-beta (TGF-beta).     

In situ PCR technique based on pricking microinjection for cDNA cloning in single cells of barley coleoptile and powdery mildew pathogen

To clone cDNAs of mRNA specifically expressed at the infection sites, we applied the polymerase chain reaction (PCR) combined with pricking microinjection to barley coleoptile epidermis inoculated with powdery mildew pathogen. In essence, first-strand cDNAs were synthesized in situ the needle-pricked epidermal cells in which fungal haustoria had formed, and were subsequently amplified by PCR with synthetic primers. The amplified DNAs were subcloned into a plasmid vector for the construction of a cDNA library. The antisense RNAs were in vitro-transcribed from subcloned DNAs, labelled, and introduced into pathogen-invaded coleoptile epidermal cells by pricking microinjection. Target cell-specific cDNAs were identified by a specific in situ hybridization in the pathogen-invaded cells. This technique was also applied to the amplification and identification of cDNAs which were reverse-transcribed from mRNAs of targeted infection structures of the powdery mildew pathogens inoculated onto barley coleoptile epidermis.     

Cloning and regulation of a mRNA specifically expressed in the preadipose state

A cDNA library of Ob1771 preadipocytes was constructed, and a cDNA clone designated pOb24 was isolated by differential screening. The pOb24 mRNA, 6 kilobases in length, rose sharply in early differentiating Ob1771 and 3T3-F442A cells and decreased thereafter. In mouse adipose tissue, it was present at a high level in stromal-vascular cells (containing adipose precursor cells) and at a low level in mature adipocytes. Thus, pOb24 mRNA appears to be both in vitro and in vivo an unique marker of the preadipose state, i.e. of cell commitment during adipose cell differentiation. In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 mRNA in Ob1771 cells required neither growth hormone or triiodothyronine as obligatory hormones nor insulin as a modulating hormone. Comparative studies of the expression of pOb24 and dihydrofolate reductase genes during the cell cycle suggest that arrest at the G1/S boundary was critical for the entry into the preadipose state. Tumor necrosis factor and transforming growth factor-beta were able to induce a large decrease of pOb24 mRNA level in growth-arrested Ob1771 cells. This decrease was shown to be only confined to early differentiating, glycerol-3-phosphate dehydrogenase negative cells as no decrease of pOb24 mRNA level was observed in glycerol-3-phosphate dehydrogenase positive cells. This result suggests that signals generated by tumor necrosis factor and transforming growth factor-beta have no effect on a commitment-related gene in late differentiated cells.     

Cloning and expression of type XII collagen isoforms during bovine adipogenesis

In order to isolate candidate genes involved in bovine adipocyte differentiation, we have constructed a subtraction library from a clonal bovine intra-muscular pre-adipocyte (BIP) cell line using the suppression subtractive hybridization method. We have isolated a set of subtracted cDNA fragments whose respective mRNA levels are up-regulated during the adipogenic differentiation of BIP cells, and cloned cDNAs from a differentiated BIP-lambda ZAP II cDNA library. Two cDNA clones were highly homologous to the sequence of mouse and human type XII collagen alpha-1, determined by a BLAST homology search. As type XII collagen has been reported to have four types of splicing isoform, two clones were determined to be XII-1 and XII-2 splicing isoforms, respectively, because of a difference in the C-terminal NC1 domain. From the expression analysis of type XII collagen, the XIIA-2 isoform was mainly expressed in differentiated BIP cells and adipose tissues. Although the function of type XII collagen has not been established as yet, these results suggest that type XII collagen may be associated with adipocyte differentiation and adipose formation in cattle and is a potentially useful marker for adipogenesis.     

OSBP-Related Proteins (ORPs) in Human Adipose Depots and Cultured Adipocytes: Evidence for Impacts on the Adipocyte Phenotype

Oxysterol-binding protein (OSBP) homologues, ORPs, are implicated in lipid homeostatic control, vesicle transport, and cell signaling. We analyzed here the quantity of ORP mRNAs in human subcutaneous (s.c.) and visceral adipose depots, as well as in the Simpson-Golabi-Behmel syndrome (SGBS) adipocyte cell model. All of the ORP mRNAs were present in the s.c and visceral adipose tissues, and the two depots shared an almost identical ORP mRNA expression pattern. SGBS adipocytes displayed a similar pattern, suggesting that the adipose tissue ORP expression pattern mainly derives from adipocytes. During SGBS cell adipogenic differentiation, ORP2, ORP3, ORP4, ORP7, and ORP8 mRNAs were down-regulated, while ORP11 was induced. To assess the impacts of ORPs on adipocyte differentiation, ORP3 and ORP8, proteins down-regulated during adipogenesis, were overexpressed in differentiating SGBS adipocytes, while ORP11, a protein induced during adipogenesis, was silenced. ORP8 overexpression resulted in reduced expression of the aP2 mRNA, while down-regulation of adiponectin and aP2 was observed in ORP11 silenced cells. Furthermore, ORP8 overexpression or silencing of ORP11 markedly decreased cellular triglyceride storage. These data identify the patterns of ORP expression in human adipose depots and SGBS adipocytes, and provide the first evidence for a functional impact of ORPs on the adipocyte phenotype.     

Up-regulation and down-regulation of genes expressed in cocultures of rat Sertoli cells and germ cells

To better understand the molecular interactions between somatic and germ cells in the mammalian testis, we have begun to analyze with mRNA differential display changes in gene expression induced by coculturing rat Sertoli cells and germ cells. We have identified 10 cDNAs that are either down-regulated or up-regulated in cocultures of germ cells and Sertoli cells. Three genes expressed in Sertoli cells and three genes expressed in germ cells were down-regulated in Sertoli cell-germ cell cocultures, whereas four genes were up-regulated in the cocultures. Northern blot analysis was used to establish the expression pattern of the mRNAs encoded by the cDNAs and to define the sizes of the differentially expressed mRNAs. Sequence analysis of the cDNAs and computer searches against the GenBank and EMBL DNA databases were used to relate the ten cDNAs to known genes. Of the three Sertoli cell cDNAs, one appeared identical to transferrin, while the other two shared regions of similarity to an endoplasmic reticulum stress protein and to a pro-α₂ XI collagen, respectively. The three germ cell cDNAs shared sequences with fibronectin, with a basic fibroblast growth factor receptor and with an IgG gamma 2b, respectively. The four cDNAs that were up-regulated in the Sertoli-germ cell cocultures showed similarity to an isoform of casein kinase 1δ, to an epidermal growth factor, to a statin-related protein, and to an integral membrane glycoprotein. These data demonstrate that a number of specific genes are up- and down-regulated when germ cells and Sertoli cells are cocultured, and suggest these genes are important in cell to cell communication during spermatogenesis. Mol. Reprod. Dev. 47:380–389, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of cDNA clones for differentially expressed genes in embryos of dormant and nondormant Avena fatua L. caryopses

The molecular regulation of seed dormancy was investigated using differential display to visualize and isolate cDNAs representing differentially expressed genes during early imbibition of dormant and nondormant Avena fatua L. embryos. Of about 3000 cDNA bands examined, 5 cDNAs hybridized with mRNAs exhibiting dormancy-associated expression patterns during the first 48 h of imbibition, while many more nondormancy-associated cDNAs were observed. Dormancy-associated clone AFD1 hybridized with a 1.5 kb mRNA barely detectable in dry dormant and nondormant embryos that became more abundant in dormant embryos after 24 h of imbibition. Clone AFD2 hybridized with two mRNAs, a 1.3 kb message constitutively expressed in dormant and nondormant embryos and a 0.9 kb message present at higher levels in dormant embryos after 3 h of imbibition. Nondormancy-associated clones AFN1, AFN2 and AFN3 hybridized with 1.5 kb, 1.7 kb and 1.1 kb mRNAs, respectively, that were more abundant in nondormant embryos during imbibition. Expression patterns of some mRNAs in dormant embryos induced to germinate by GA₃ treatment were different than water controls, but were not identical to those observed in nondormant embryos. DNA sequence analysis revealed 76% sequence identity between clone AFN3 and a Citrus sinensis glutathione peroxidase-like cDNA, while significant sequence similarities with known genes were not found for other clones. Southern hybridization analyses showed that all clones represent low (1 to 4) copy number genes.     

Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.     

Isolation of 10 differentially expressed cDNAs in differentiated Neuro2a cells induced through controlled expression of the GD3 synthase gene

Recently, we showed that transfection of GD3 synthase cDNA into Neuro2a cells, a mouse neuroblastoma cell line, causes cell differentiation with neurite sprouting. In a search for the genes involved in this ganglioside-induced Neuro2a differentiation, we used a tetracycline-regulated GD3 synthase cDNA expression system combined with differential display PCRs to identify mRNAs that were differentially expressed at four representative time points during the process. We report here the identification of 10 mRNAs that are expressed highly at the Neuro2a differentiated stage. These cDNAs were named GDAP1-GDAP10 for (ganglioside-induced differentiation-associated protein) cDNAs. It is interesting that in retinoic acid-induced neural differentiated mouse embryonic carcinoma P19 cells, GDAP mRNA expression levels were also up-regulated (except that of GDAP3), ranging from three to >10 times compared with nondifferentiated P19 cells. All the GDAP genes (except that of GDAP3) were developmentally regulated. The GDAP1, 2, 6, 8, and 10 mRNAs were expressed highly in the adult mouse brain, whereas all the other GDAP mRNAs were expressed in most tissues. Our results suggested that these GDAP genes might be involved in the signal transduction pathway that is triggered through the expression of a single sialyltransferase gene to induce neurite-like differentiation of Neuro2a cells.