Regulated Gene Family Research Articles

Bifidobacterium longum supplementation improved high-fat-fed-induced metabolic syndrome and promoted intestinal Reg I gene expression

Recent evidence suggests that intestinal Bifidobacterium species (spp.) positively correlates with improved insulin resistance and obesity, and this might be linked to metabolic inflammation. The expression of intestinal REG (regenerating) family proteins which are widely involved in inflammatory bowel disease and diabetes are still unknown in metabolic syndrome. Hence, we investigated the effects of Bifidobacterium longum (BIF) supplementation on metabolic parameters, intestinal function and expression of Reg family genes in a rat model of metabolic syndrome induced by a high-fat (HF) diet. We specifically increased the gut bifidobacterial content of HF-fed rats through BIF supplementation. Compared with the normal chow-fed control rats, HF feeding significantly reduced intestinal Bifidobacterium. As expected, BIF supplementation fed rats had totally restored quantities of Bifidobacterium. HF diet-fed rats showed significant increase in body weight, fat deposits, systolic blood pressure, fasting glucose, fasting triglycerides and reduced insulin sensitivity, while increases of intestinal Bifidobacterium did improve HF-diet-induced metabolic disorders. HF feeding led to significantly higher levels of the plasma lipopolysaccharide, interleukin-1β and intestinal myeloperoxidase, as well as intestinal inflammatory activity index, while these parameters were normalized to the control levels in the HF + BIF-treated rats. The levels of RegI mRNA and protein in the HF + BIF group were significantly higher than the control and the HF groups. Increasing Bifidobacterium in the gut improved HF-fed-induced metabolic syndrome by reducing metabolic endotoxin concentrations and intestinal inflammation, as well as upgrading the expression of intestinal Reg I as a regulator of growth factor.

Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of cotton ( Gossypium hirsutum L.)

High salinity is one of the main factors limiting cotton growth and productivity. The genes that regulate salt stress in TM-1 upland cotton were monitored using microarray and real-time PCR (RT-PCR) with samples taken from roots. Microarray analysis showed that 1503 probe sets were up-regulated and 1490 probe sets were down-regulated in plants exposed for 3 h to 100 mM NaCl, and RT-PCR analysis validated 42 relevant/related genes. The distribution of enriched gene ontology terms showed such important processes as the response to water stress and pathways of hormone metabolism and signal transduction were induced by the NaCl treatment. Some key regulatory gene families involved in abiotic and biotic sources of stress such as WRKY, ERF, and JAZ were differentially expressed. Our transcriptome analysis might provide some useful insights into salt-mediated signal transduction pathways in cotton and offer a number of candidate genes as potential markers of tolerance to salt stress.

The transcriptome landscape of Arabidopsis male meiocytes from high‐throughput sequencing: the complexity and evolution of the meiotic process

Meiosis is essential for eukaryotic sexual reproduction, with two consecutive rounds of nuclear divisions, allowing production of haploid gametes. Information regarding the meiotic transcriptome should provide valuable clues about global expression patterns and detailed gene activities. Here we used RNA sequencing to explore the transcriptome of a single plant cell type, the Arabidopsis male meiocyte, detecting the expression of approximately 20 000 genes. Transcription of introns of >400 genes was observed, suggesting previously unannotated exons. More than 800 genes may be preferentially expressed in meiocytes, including known meiotic genes. Of the 3378 Pfam gene families in the Arabidopsis genome, 3265 matched meiocyte-expressed genes, and 18 gene families were over-represented in male meiocytes, including transcription factor and other regulatory gene families. Expression was detected for many genes thought to encode meiosis-related proteins, including MutS homologs (MSHs), kinesins and ATPases. We identified more than 1000 orthologous gene clusters that are also expressed in meiotic cells of mouse and fission yeast, including 503 single-copy genes across the three organisms, with a greater number of gene clusters shared between Arabidopsis and mouse than either share with yeast. Interestingly, approximately 5% transposable element genes were apparently transcribed in male meiocytes, with a positive correlation to the transcription of neighboring genes. In summary, our RNA-Seq transcriptome data provide an overview of gene expression in male meiocytes and invaluable information for future functional studies.

Reg4 protects against acinar cell necrosis in experimental pancreatitis

Background and aimsReg4 is a recently discovered member of the regenerating gene family with distinctive expression profiles in primary cancers. To date, the physiological function of Reg4 is poorly understood....

Reg IV protein and mRNA expression in different rat organs

The Reg IV gene has been documented in the human colon, small intestine, stomach and pancreas. Expression of the Reg IV in different cell types has been associated with regeneration, cell growth and cell survival, cell adhesion and resistance to apoptosis. Since the distribution of the Reg IV protein in normal rat tissues is unknown, the aim of this study was to reveal the expression of the Reg IV protein in structurally and functionally different rat organs. The expression of Reg IV gene was analyzed by Western blot and reverse transcription-polymerase chain reaction. Immunohistochemistry was used to localize Reg IV protein. Reg IV protein was expressed in pancreas, stomach, small intestine, colon, brain, spleen, kidney and urinary bladder in two-month-old male Wistar rats. In addition, the expression of Reg IV mRNA by reverse transcription-polymerase chain reaction was confirmed. Our study provides detailed information about the expression and localization of Reg IV protein in different rat organs. These findings provide an evidence of Reg IV expression in different rat organs, which may help elucidate a potential role in growth and proliferation of different cells like other members of the Reg family genes which act as growth factors in the different organs.

Increased NDRG1 Expression is Associated with Advanced T Stages and Poor Vascularization in Non-small Cell Lung Cancer

N-myc downstream regulated gene 1 (NDRG1) is a member of the N-myc downstream regulated gene family which belongs to the alpha/beta hydrolase superfamily. Earlier studies have shown its association with inhibition of tumor metastasis. However, its function in malignant tumors is not fully enunciated. Recently there was increasing evidence that NDRG1 is involved in stress responses. In the current study, we examined the expression of NDRG1 and its correlation with clinicopathological factors and microvessel density (MVD) in non-small cell lung cancer (NSCLC) using immunohistochemistry (IHC). NDRG1 expression in NSCLC (71/115, 61.7%) was higher than that in normal lung tissues (32/115, 27.8%) (p < 0.05). NDRG1 expression in NSCLC cells was found in cytoplasm (63/115, 54.8%), nuclear (24/115, 20.9%) and cell membrane (13/115, 11.3%). NDRG1 expression in NSCLC with advanced T stages (T2-4) (63/84, 75.0%) was significantly higher than that with T1 stage (8/31, 25.8%) (P < 0.05). No other clinicopathological factors including lymph node metastasis were found to be associated with NDRG1 expression (p > 0.05). Moreover increased NDRG1 expression was associated with lower MVD in NSCLC (P < 0.05). MVD in adenocarcinoma (33.4 ± 8.4/HP) was significantly higher than that in squamous cell carcinoma (SCC) (19.3 ± 8.1/HP) (P < 0.05). No other clinicopathological factors were associated with MVD in NSCLC (p > 0.05). The present findings indicate an increase of NDRG1 expression with the progress of tumour extent which may be due to unbalanced tumor oxygenation on account of poor vascularization in NSCLC.

RegIV expression showing specificity to gastrointestinal tract and its potential role in diagnosing digestive tract neuroendocrine tumor

Regenerating gene IV (RegIV), a member of the regenerating gene family discovered in 2001, has been found to be involved in malignancy in several different organs including the stomach, colorectum, pancreas and prostate, but the overall expression profile of RegIV has not been reported. To learn more about RegIV, we evaluated its distribution by immunohistochemistry (IHC) in a total of 360 samples including 24 types of normal tissue, 40 benign and malignant lesions, and 18 neuroendocrine tumors. We found that in normal tissues, in addition to its relative specificity for the gastrointestinal tract, RegIV was detected in the adrenal gland and mammary gland. Among all the malignancies of various histological types under evaluation, RegIV was found mostly in adenocarcinomas. Studies on additional sets of colorectal tumor samples showed that RegIV expression was predominant in colorectal adenoma (87.5%) and peritumoral tissue (100%) but not in cancer tissue (30.8%). Among neuroendocrine tumors, RegIV had a relatively restricted expression to those of digestive system.

REG4 acts as a mitogenic, motility and pro-invasive factor for colon cancer cells

REG4, the latest member of the regenerating gene family, is overexpressed in inflammatory bowel diseases and gastrointestinal carcinomas. To date, its pathophysiologic role has not been well established. Using HT-29 models, we previously identified REG4 as being overexpressed in colorectal tumor cells displaying a drug-resistance phenotype; some also displayed invasive properties. Thus, we investigated the potential functions of REG4 in biological processes involved in colorectal tumor progression such as cell proliferation, migration and invasion. Colon cancer cells secreting REG4 (HT29-5M21, HT29-5F7 and HT29/REG4-8) or not (HT-29, HT29/CT1 and Caco-2/TC7) were used to analyze the autocrine and paracrine effects of REG4. REG4 was continuously secreted into the culture medium of colon cancer cells. REG4 stimulated cell growth in a paracrine manner after 24 h of treatment. Notably, REG4 promoted migration and invasion of tumor cells in both an autocrine and paracrine manner, and these effects were significantly decreased by concomitant treatment with an anti-REG4 antibody. Using pharmacological inhibitors, we showed that PI3K/Akt, PKAs, PKCs and Rho-like GTPases, but not MAPK, are involved in REG4 invasion signals. In addition, REG4 expression was found to be increased in tissues harboring proliferation and migration properties such as the developing intestine and tissues from inflammatory bowel disease, hyperplastic polyps, adenoma and colorectal cancers. In various situations, REG4 expression was not confined to proliferating cells, regenerating cells or cells of the invasive front of metastatic tumors, suggesting that extracellular REG4 may act on epithelial cells in a paracrine manner. Altogether, our results indicate that REG4 is a multifunctional secreted protein which acts on colorectal cancer cells in an autocrine and paracrine manner. According to its biological functions and tissue expression, REG4 may play an important role in the development and progression of colorectal cancer, as well as in intestinal morphogenesis and epithelium restitution.

LFG: a candidate apoptosis regulatory gene family

The expanding wealth of human, model and other organism's genomic data has allowed the identification of a distinct gene family of apoptotic related genes. Most of these genes are currently unannotated or have been subsumed under two questionably related gene families in the past. For example the transmembrane Bax inhibitor 1 (BI1) motif family has been reported to play a role in apoptosis and to consist of at least seven mammalian protein genes, GRINA, BI1, Lfg/FAIM2, Ghitm, RESC1/Tmbim1, GAAP/Tmbim4, and Tmbm1b. However, a detailed sequence and phylogenetic analysis shows that only five of these form a clear and unique protein family. This now provides information for understanding and investigating the biological roles of these proteins across a wide range of tissues in model organisms. The evolutionary relationships among these genes provide a powerful prospective for extrapolating to human conditions.

Purification of a bioactive recombinant human Reg IV expressed in Escherichia coli

Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily. Reg IV is highly expressed in the gastrointestinal tract and markedly up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel disease. However, the physiological and pathological functions of Reg IV are largely unknown, partly due to the limited access of the bioactive protein. We report here the first expression and purification of Reg IV proteins using a prokaryotic system. Human Reg IV was expressed in Escherichia coli as an insoluble protein which was identified in the fraction of inclusion body after ultrasonication of the bacteria. After the protein aggregate was solubilized by guanidine–HCl, it was refolded by sucrose and arginine-assisted procedures and purified using cation-exchange chromatography. The protein identity and purity of the final preparation were confirmed by analysis of the protein mass and immune specificity in SDS–PAGE, Western blotting, and HPLC assay. The biological activity of the protein was determined by the HCT116 and HT29 cell proliferation assays. The highly purified bioactive human Reg IV should aid in further characterization of its physiological and pathological functions.

Expression profile of REG family proteins REG Iα and REG IV in advanced gastric cancer: comparison with mucin phenotype and prognostic markers

Regenerating gene family members 1 (REG Iα) and 4 (REG IV) are overexpressed in a subset of gastric cancers. However, comparative characterization of the expression of these family proteins has remained unclear. Therefore, we aimed to elucidate not only the association between REG protein expression and mucin phenotype but also their significance as a prognostic marker for patients with gastric cancer. The expression of REG Iα, REG IV, CDX2, MUC2, and MUC5AC in gastric cancer tissues was examined by immunohistochemistry. The relationship between REG protein expression and clinicopathological parameters or mucin phenotype was then analyzed. REG Iα and REG IV expression was positive in 33 (52%) and 31 (49%) of 63 gastric cancers examined, respectively. REG Iα expression was significantly related to venous invasion and tumor stage, whereas REG IV expression showed no relationship to clinicopathological features. With regard to mucin phenotype, REG IV expression was significantly correlated with MUC2 and CDX2 expression, suggesting an association with the intestinal mucin phenotype of gastric cancer. On the other hand, REG Iα expression had no correlation with MUC2, CDX2, or MUC5AC in gastric cancer tissues. Expression of REG Iα but not REG IV was an independent predictor of poor outcome in patients with gastric cancer. In addition, patients with gastric cancer negative for both REG Iα and REG IV expression had a significantly better outcome than patients positive for either REG Iα or REG IV. Profiling of REG protein expression is useful to for prognostication of patients with gastric cancer.

Photoperiod insensitive Ppd-A1a mutations in tetraploid wheat (Triticum durum Desf.)

Variation in photoperiod response plays an important role in adapting crops to agricultural environments. In hexaploid wheat, mutations conferring photoperiod insensitivity (flowering after a similar time in short or long days) have been mapped on the 2B (Ppd-B1) and 2D (Ppd-D1) chromosomes in colinear positions to the 2H Ppd-H1 gene of barley. No A genome mutation is known. On the D genome, photoperiod insensitivity is likely to be caused by deletion of a regulatory region that causes misexpression of a member of the pseudo-response regulator (PRR) gene family and activation of the photoperiod pathway irrespective of day length. Photoperiod insensitivity in tetraploid (durum) wheat is less characterized. We compared pairs of near-isogenic lines that differ in photoperiod response and showed that photoperiod insensitivity is associated with two independent deletions of the A genome PRR gene that cause altered expression. This is associated with induction of the floral regulator FT. The A genome deletions and the previously described D genome deletion of hexaploid wheat remove a common region, suggesting a shared mechanism for photoperiod insensitivity. The identification of the A genome mutations will allow characterization of durum wheat germplasm and the construction of genotypes with novel combinations of photoperiod insensitive alleles.

RANKL-induced schlafen2 is a positive regulator of osteoclastogenesis

Osteoclasts are hematopoietic lineage derived-multinucleated cells that resorb bone. Their activity in balance with that of osteoblast is essential for bone homeostasis. Receptor activator of NF-κB ligand (RANKL) is known as an essential cytokine for the osteoclastogenesis, and c-Jun signaling in cooperation with NFAT family is crucial for RANKL-regulated osteoclastogenesis. We show here that schlafen2 (Slfn2), a member of a new family of growth regulatory genes involved in thymocyte development, is critical for osteoclastogenesis. RANKL selectively induces Slfn2 expression in osteoclast precursors via Rac1 signaling pathway. Targeted inhibition of Slfn2 by small interfering RNAs (siRNAs) markedly inhibits the formation of osteoclasts by diminishing the activation of c-Jun and the expression of c-Jun and NFATc1. In contrast, the overexpression of Slfn2 markedly increased phosphorylation and transactivation of c-Jun by RANKL. Together, these results indicate that Slfn2 has an essential role in osteoclastogenesis, functioning upstream of c-Jun and NFATc1.

Gene expression profiles of cardiomyocytes in rat autoimmune myocarditis by DNA microarray and increase of regenerating gene family

Cardiomyocytes with myocarditis compared with the normal state are thought to change the expressions of various genes greatly, some of which may be new biomarkers or new biologic medicinal products. However, until now, little comprehensive analysis has been made of gene-expression changes in cardiomyocytes with myocarditis. In this study, we performed a DNA microarray analysis by using cardiomyocytes from rat experimental autoimmune myocarditis (EAM). On day 0, rats were immunized with porcine cardiac myosin and cardiomyocytes were isolated and purified from EAM hearts and normal hearts by a method that is hardly thought to change gene expressions in cardiomyocytes. RNA from normal cardiomyocytes and cardiomyocytes of EAM on day 18 was analyzed for 7711 gene expressions by DNA microarray. Some gene expressions showed over 10-fold changes. In particular, the regenerated gene (Reg)2/pancreatitis-associated protein (PAP)1 messenger RNA (mRNA) level most markedly increased in the genes, which were clearly expressed in cardiomyocytes rather than in noncardiomyocytes, and it was approximately 2000-fold greater in cardiomyocytes under active myocarditis than normal by real-time reverse transcription polymerase chain reaction analysis. Moreover, we demonstrated that Reg2/PAP1 proteins determined by Western blot analysis and immunohistochemistry and other Reg/PAP family gene expressions were remarkably increased in EAM hearts; in addition, interleukin (IL)-6 expression was significantly related to Reg2/PAP1. It seemed that these data were useful as a reference database of gene-expression changes in cardiomyocytes with myocarditis. The Reg/PAP family, which was found to show dramatically increasing gene expressions by DNA microarray analysis, was suspected to play an important role in myocarditis.

Advances in regenerating gene family and digestive system neoplasms

Advances in regenerating gene family and digestive system neoplasms

A Positive Role for NLI/Ldb1 in Long-Range β-Globin Locus Control Region Function

Long-range interactions between distant regulatory elements, such as enhancers, and their target genes underlie the specificity of gene expression in many developmentally regulated gene families. NLI/Ldb1, a widely expressed nuclear factor, is a potential mediator of long-range interactions. Here, we show that NLI/Ldb1 and erythroid-binding partners GATA-1/SCL/LMO2 bind in vivo to the beta-globin locus control region (LCR). The C-terminal LIM interaction domain of NLI is required for formation of the complex on chromatin. Loss of the LIM domain converts NLI into a dominant-negative inhibitor of globin gene expression, and knockdown of NLI by using shRNA results in failure to activate beta-globin expression. Kinetic studies reveal that the NLI/GATA-1/SCL/LMO2 complex is detected at the beta-globin promoter coincident with RNA Pol II recruitment, beta-globin transcription, and chromatin loop formation during erythroid differentiation, providing evidence that NLI facilitates long-range gene activation.

Transcript profiles of the cytokinin response regulator gene family in Populus imply diverse roles in plant development

Cytokinins are plant hormones that influence diverse processes of growth and development. In this study the cytokinin response regulators (RRs) were identified, annotated and characterized at the transcript level in Populus balsamifera ssp. trichocarpa genotype Nisqually 1. The Populus genome was searched for genes that exhibit high sequence identity across their receiver domains. Gene structure was determined by prediction software and, where possible, corroborated by publicly available expressed sequence tags (ESTs). Thirty-three genes belonging to the cytokinin RR gene family were identified in Populus: 11 type As, 11 type Bs and 11 pseudo-RRs. Developmental and cytokinin-responsive expression of the Populus RRs was assessed by whole-genome microarrays and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Populus RR type As and type Bs appear to be preferentially expressed in nodes, while pseudo-RRs are preferentially expressed in mature leaves. Seven type As and three type Bs were rapidly induced by exogenous cytokinin. Organ-preferred expression patterns suggest possible roles for type As and Bs in development and for pseudo-RRs in integration of environmental signals with plant function.

Quantitative expression of the homeobox and integrin genes in human gastric carcinoma

The homeobox (HOX) genes are a large family of regulator genes involved in the control of developmental processes and cell differentiation. The HOX genes encode transcription factors, and an increasing number of studies have shown that these genes may be implicated in the growth and the progression of many types of tumours. The present study investigated the expression of the HOX and integrin genes and their relationships in gastric carcinoma. We analyzed the RNA expression of 13 HOX genes from HOXA, C and D clusters and alphaV, alpha5 and alpha8 integrin genes in 24 gastric cancer samples by quantitative real-time PCR. The results showed that the HOXA2 gene and the alpha8 integrin gene had a lower expression in tumour samples than in normal gastric mucosas. The comparison between the HOX and integrin genes showed that HOXA2 and alphaV integrin expression presented the same trend in 83% of the samples. Moreover, in cancer samples that expressed the HOXD11 gene, the expression of alphaV integrin was lower with respect to normal mucosas. The different roles of HOX and integrin genes in gastric carcinoma remain to be fully elucidated. These findings suggest that the HOX genes may play a critical role in the genesis, maintenance and diffusion of gastric carcinoma.

Transcription Factors GATA-1 and Fli-1 Regulate Human HOXA10 Expression in Megakaryocytic Cells

HOXA10 is a member of the HOX family of regulatory genes that are involved in hematopoiesis. Its role in megakaryopoiesis has been suggested by its expression in immature megakaryocytes and by the proliferation of megakaryocyte-primitive blast colonies upon HOXA10 overexpression. We sought to understand the role of HOXA10 in megakaryopoiesis better, by investigating its transcriptional regulation. Analysis of the 5' untranslated region and transfection of promoter/plasmids into human tissue culture cell lines identified transcriptionally active sequences that contain GATA-1 and Ets-1 sites and a putative binding site for its neighboring gene, HOXA11. Gel shift assays confirmed protein-DNA interactions at these sites. Mutation of the GATA-1 and the Ets-1 motifs amplified the expression of HOXA10 in HEL and K562 cells, confirming the importance of these cis-acting elements in regulating HOXA10 expression in megakaryocytic cells. Chromatin immunoprecipitation (ChIP) and chloramphenicol acetyl transferase (CAT) assays confirm that HOXA11 binds to the putative binding site, resulting in repression of HOXA10 expression. These data taken together give insight into the regulation of HOXA10 expression in megakaryocytic differentiation.

A Pseudo-Response Regulator is misexpressed in the photoperiod insensitive Ppd-D1a mutant of wheat (Triticum aestivum L.)

Ppd-D1 on chromosome 2D is the major photoperiod response locus in hexaploid wheat (Triticum aestivum). A semi-dominant mutation widely used in the "green revolution" converts wheat from a long day (LD) to a photoperiod insensitive (day neutral) plant, providing adaptation to a broad range of environments. Comparative mapping shows Ppd-D1 to be colinear with the Ppd-H1 gene of barley (Hordeum vulgare) which is a member of the pseudo-response regulator (PRR) gene family. To investigate the relationship between wheat and barley photoperiod genes we isolated homologues of Ppd-H1 from a 'Chinese Spring' wheat BAC library and compared them to sequences from other wheat varieties with known Ppd alleles. Varieties with the photoperiod insensitive Ppd-D1a allele which causes early flowering in short (SD) or LDs had a 2 kb deletion upstream of the coding region. This was associated with misexpression of the 2D PRR gene and expression of the key floral regulator FT in SDs, showing that photoperiod insensitivity is due to activation of a known photoperiod pathway irrespective of day length. Five Ppd-D1 alleles were found but only the 2 kb deletion was associated with photoperiod insensitivity. Photoperiod insensitivity can also be conferred by mutation at a homoeologous locus on chromosome 2B (Ppd-B1). No candidate mutation was found in the 2B PRR gene but polymorphism within the 2B PRR gene cosegregated with the Ppd-B1 locus in a doubled haploid population, suggesting that insensitivity on 2B is due to a mutation outside the sequenced region or to a closely linked gene.