Acid-inducible Gene Research Articles

Retinoic acid-inducible G protein-coupled receptors bind to frizzled receptors and may activate non-canonical Wnt signaling

Frizzled (Fz) seven-pass transmembrane receptors are Wnt receptors and function in a variety of developmental pathways. Here we identify retinoic acid-inducible gene-1, 2, 3, and 4 (RAIG1, 2, 3, and 4) as potential Fz binding proteins. RAIG proteins are seven-pass transmembrane receptors, and Xenopus RAIG2, 3, and 4 are expressed in early gastrula. XRAIG2 can activate small GTPases, such as RhoA, Rac, and Cdc42, and c-jun N-terminal kinase, thus exhibit activities that overlap with non-canonical Wnt/Fz signaling. Injection of XRAIG2 mRNA into Xenopus embryo causes a severe shortened and bent body axis due to defective gastrulation movements, reminiscent of abnormal non-canonical Wnt signaling. XRAIG2 affects convergent extension in activin-treated animal caps, which can be partially rescued by co-injection of a dominant-negative form of Cdc42. In zebrafish embryo, XRAIG2 also causes Ca 2+ flux, one of the consequences of non-canonical Wnt signaling. These results suggest a possible crosstalk/integration between Wnt/Frizzled and RAIG signal transduction pathways.

JWA Gene is Involved in Cadmium-induced Growth Inhibition and Apoptosis in HEK-293T Cells

Cadmium (Cd) is widely dispersed in the environment due to occupational and personal (cigarette) emissions. Exposure of human embryonic kidney 293T (HEK-293T) cells to CdCl2 resulted in growth inhibition and apoptosis. Our previous studies demonstrated that JWA, a novel retinoic acid-inducible and cytoskeleton-associated gene, is a potential environmental-responsive gene with increased expression attributed to oxidative and heat-shock stresses. In the present study, JWA was also found to be responsive to Cd exposure. After treatment with 20 μM CdCl2 for 12 h, the expression level of JWA was increased with accompanied growth inhibition and apoptosis. In addition, knock-down JWA protein expression by using transient transfecting of HEK-293T cells with antisense JWA express vector showed a protective effect against Cd-induced apoptosis. To determine whether the upregulation of JWA by Cd involved regulation by transcriptional mechanisms, further reporter gene assays were employed, which demonstrated a marked increase in JWA promoter activity. In addition, elevated intracellular levels of ROS components (O2 − · and H2O2) and activation of JNK, ERK, and MAPK were found with corresponding upregulation of JWA protein expression. These results suggest that Cd-induced growth inhibition and apoptosis may involve ROS generation and subsequent affect on MAPK signal pathway. JWA responsiveness to CdCl2 might be through both transcriptional and posttranslational mechanisms.

JWA—A Novel Environmental-Responsive Gene, Involved in Estrogen Receptor-Associated Signal Pathway in MCF-7 and MDA-MB-231 Breast Carcinoma Cells

The pyrethroid insecticide fenvalerate and the organophosphorus insecticide phoxim are now the most widely used agents for indoor pest control in China. Fenvalerate was shown to mimic estrogenic activity, whereas phoxim did not induce similar effects. Our previous studies demonstrated that JWA, a novel retinoic acid-inducible and cytoskeleton-associated gene, is also a potential environmental-responsive gene with increased expression to oxidative and heat-shock stresses. In the present study, the influence of both fenvalerate and phoxim was examined on the expression of JWA in MCF-7 (ER+) and MDA-MB-231 (ER−) human breast carcinoma cell lines. Concentrations of 0.01, 1, and 100 μmol/L of fenvalerate or phoxim were selected to treat both MCF-7 and MDA-MB-231 cells at 1, 3, and 5 d, respectively. The MTT results only showed that fenvalerate stimulated MCF-7 cell proliferation. Western blot assay was employed to detect the expressions of JWA and heat-shock proteins (hsp27 and hasp70). The results showed that after treatment with fenvalerate, both JWA and hsp70 showed similar expression patterns in the both cell lines; however, all the expression patterns of JWA, hsp27, and hsp70 were evidently reversed between ER+ and ER− cells. In addition, phoxim-treated cells showed a concentration-dependent relationship in JWA expression at all time points. These results suggest that JWA has similar functions with respect to hsp27 and hsp70, and might be a novel signal molecule in estrogen receptor-related signal transduction pathways in mammalian cells.This work was partially supported by the following grants: National Natural Science Foundation of China (30170812), the Foundation of High-tech Key Project, Education Department, Jiangsu Province (JH01-049), the National Key Basic Research and Development Project (973) (grant 2002CB512900), and the Project of the Ministry of Science and Technology of China (grants 2001-50 and 2000-026).

Transcriptional analysis of the acid-inducible asr gene in enterobacteria

We show here that transcription of the asr gene in Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae and Enterobacter cloacae is strongly dependent on the acidification level of the growth medium, with maximal induction at pH 4.0–4.5 as determined by Northern hybridization analysis. Previous gene array analyses have also shown that asr is the most acid-induced gene in the E. coli genome. Sequence alignment of the asr promoters from different enterobacterial species identified a highly conserved region located at position −70 to −30 relative to the asr transcriptional start site. By deletion of various segments of this region in the E. coli asr promoter it was shown that sequences upstream from the −40 position were important for induction. Transcription from the E. coli asr promoter was demonstrated to be growth-phase-dependent and to require the alternative sigma factor RpoS (σ S) in stationary phase. Transcription of the asr gene was also found to be subject to negative control by the nucleoid protein HNS.

The RAIG Family Member, GPRC5D, Is Associated with Hard-Keratinized Structures

Retinoic acid-inducible gene-1 was originally identified as an orphan G-protein coupled receptor induced by retinoic acid. Three highly homologous oGPCR (GPRC5B, GPRC5C, and GPRC5D) have since been classified into the RAIG1 family. We describe here, the unique tissue distribution of GPRC5D and its mechanism of expression. Hybridization in situ has shown that GPRC5D is expressed in differentiating cells that produce hard keratin, including cortical cells of the hair shaft, the keratogenous zone of the nail, and in a central region of the filiform papillae of the tongue. The GPRC5D transcript is expressed in hair follicles during mid- and late anagen, and catagen but not at telogen and early anagen phases. The differentiation-inducer, all-trans retinoic acid, induces GPRC5D expression in cultured hair bulb cells. Because the tissue distribution of GPRC5D indicates a relationship with hard keratins that constitute the major structural proteins of hard epithelial tissues, we investigated the effect of GPRC5D on acid hard keratins. Analyses of cultured cells showed that transient overexpression resulted in suppression of Ha3 and stimulation of Ha4 hair keratin gene expression. The expression was maintained in the hair follicles of whn-deficient (nude) mice, suggesting that this gene is regulated by a signal pathway different from that of hair keratin synthesis. Collectively, these data provide a framework for understanding the molecular mechanisms of GPRC5D function in hard keratinization.

Molecular Cloning and Characterization of Two Novel Retinoic Acid-Inducible Orphan G-Protein-Coupled Receptors (GPRC5B and GPRC5C)

Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABAB receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative TaqMan PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405–D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352–D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.

Glucose modulates the abscisic acid-inducible Rab16A gene in cereal embryos.

Glucose effects on the expression of the abscisic acid-inducible Rab16A gene were examined in rice and barley embryos. Glucose feeding to rice embryos negatively affects the endogenous abscisic acid content and represses the promoter activity of the Rab16A gene. Glucose repression of the Rab16A gene takes place both at a transcriptional and a post-transcriptional level. Modulation of the abscisic acid content in rice embryos triggered by glucose did not directly influence the expression of the rice alpha-amylase gene RAmy3D, which is known to be under glucose control. The possible interaction between the glucose and abscisic acid signaling pathway is discussed.

Effect of promoter sequence on transient expression of the β-glucuronidase gene in embryogenic calli of Larix × eurolepis and Picea mariana following microprojection

The transient expression of the β-glucuronidase reporter gene was compared in embryogenic cell lines of Larix × eurolepis (L. decidua × L. leptolepis) and Picea mariana after introduction of eight vectors containing different promoter sequences using the Dupont Biolistic™ particle delivery system. Transient β-glucuronidase gene expression was highest in cells of both species after bombardment using the wheat abscisic acid inducible Em gene promoter. Transient β-glucuronidase gene expression was comparable in P. mariana and L. × eurolepis for all vectors, with the exception of the rice actin promoter that yielded higher activity in P. mariana than in L. × eurolepis. The Em gene promoter proved inducible by abscisic acid; upon the addition of abscisic acid to the culture medium, β-glucuronidase gene expression was increased 2.3- and 4.4-fold for L. × eurolepis and P. mariana, respectively. Investigation of β-glucuronidase gene expression over time showed that all transient activity disappeared 16 days after microprojection. Key words: Larix, Picea, transformation, β-glucuronidase, tissue cultures.