THE GLUCOCORTICOID-CONTROLLED GENE REGULATORY REGIONS DATABASE (GR-TRRD): THE USING FOR THE ELUCIDATION OF THE MECHANISMS OF CHEMICAL HEPATOCARCINOGENS ACTION

MERKULOVA T.I.+, MERKLOV V.M., KROPATCHEV K.Y., KALEDIN V.I.

Institute of Cytology and Genetics, (Siberian Branch of the Russian Academy of Sciences), 10 Lavrentieva ave., Novosibirsk, 630090 Russia

e-mail:merk@cgi.nsk.su

+Corresponding author

Keywords: glucorticoid-controlled gene, regilatory region, mechanism, hepatocarcirogens action, HNF3, DNA-binding activity, o-aminoazotoluene

 

The database GR-TRRD compiling the data on regulatory regions of 39 glucocorticoid-controlled genes is described. The database contains the description of over 80 glucocorticoid response elements (GREs) and 150 other regulatory elements, involved in tissue-specific, development and environmental regulation of glucocorticoid controlled genes. Comparative analysis of the regulatory regions of genes coding for tyrosine aminotransferase, aspartate aminotransferase and phosphoenolpyruvate carboxykinase allowed to propose that hepatocyte nuclear factor 3 (HNF3) is the likely target for hepatocarcinogen o-aminoazotoluene (ОАТ). This prediction has been confirmed by gel retardation experiments.

Introduction

Glucocorticoids influence a vast number of complex physiological and pathological processes. They help regulate protein, fat and carbohydrate metabolism, participate in immunosupression and anti-inflammation and are important for the optimum functioning of the cardiovascular, kidney, and nervous system. Glucocorticoids bring about their multiple effects by activating the intracellular glucocorticoid receptor (GR) that binds to specific glucocorticoid responsive elements (GREs) in the vicinity of regulated genes and subsequently affect their expression. For systematization, generalization, and analysis of constantly growing amount of experimental data on the glucocorticoid-controlled genes, the GR-TRRD (Glucocorticoid Regulated genes – Transcription Regulatory Regions Database) has been created as a constituent of the TRRD database [Kel’ A.E. et al., 1997]. The GR-TRRD is accessible at http://wwwicg.bionet.nsc.ru/trrd/34/gluc.htm.

Contents of the GR-TRRD database

The current release of the GR-TRRD (Release 4.0) compiles the data on 39 glucocorticoid-controlled genes (Table 1), including genes coding for enzymes of carbohydrate and amino acids metabolism (5), hormones(7) and hormone receptors(3), acute-phase proteins(7), serum(4), milk proteins(3), viral genes and others. Among them, 8 genes are of human origin; 18, rat; 4, mouse; 1, rabbit; 1, frog; 1, bovine; and 1, chicken. GR-TRRD contains the description of structural-functional organization of transcription regulatory regions of these genes, including the description of over 80 glucocorticoid response elements (GREs) and 150 other regulatory elements, involved in tissue-specific, development and environmental regulation of glucocorticoid controlled genes. An important component of GR-TRRD as a constituent of the TRRD database is RE field where the specificity of gene expression and regulation is described.

Transcription factor HNF3 is a target for hepatocarcinogen o-aminoazotoluene

Diminished glucocorticoid induction of tyrosine aminotransferase (ТАТ, G000798) gene expression is a well-known early effect of many hepatocarcinogens [Miller M.S.&Vogan G.N., 1986]. Meanwhile, it was shown that carcinogens do not reduce the glucocortiocoid inducibility of some other hepatic enzymes controlled by these hormones : aspartate aminotransferase (ASPART, G001175) and phosphoenolpyruvate carboxykinase(PEPCK, G000782) [Yeoh G.C.T, 1981 ]. So transcription factors that are involved in glucocorticoid regulation of the genes coding for TAT but not ASPART and PEPCK appear to be likely targets for carcinogen action. Comparative analysis of the regulatory regions of these genes described in the GR-TRRD allowed to propose that such are members of the HNF3 and Ets families [Merkulova T.I. et al., 1997] and GME-binding proteins [Oshima H. et al., 1995]. The most likely candidates are HNF3 family members, since TAT is expressed and regulated by glucocorticoids only in liver, while ASPART и PEPCK are also glucocorticoid induced in kidney, where HNF3 factors are virtually absent (G001244, G001010, G001242, G001172, G001240) (RE fields in corresponding entries of GR-TRRD).

For that reason we assessed the DNA-binding activity of these factors in nuclear extracts from the livers of intact mice and those treated with the hepatocarcinogen o-aminoazotoluene (ОАТ). The experimental animal strains were ones with different sensitivity to liver tumor induction with OAT [Kaledin V.I.& Zakharova N.P.,1984]. DNA-binding activity of the AP1 family proteins, which can act, depending on the situation, either antagonistic to or synergistic with the glucocorticoid receptor [Merkulova T.I. et al., 1997], was measured as well. Gel retardation experiments demonstrated, that after OAT administration HNF3 DNA-binding activity was strongly reduced in the nuclear extracts from the livers of sensitive A and SWR mice, which have liver tumors induced with a high frequency (70-100%). Much less effect was found in the insensitive CC57Br and AKR strains, in which case induced tumours are rare (0-6%) (Fig.1A and the summary of the quantitative results in Figure 2B). The DNA-binding activity of Ets, AP1 factors and GME-binding proteins is not affected by OAT injection. The use of specific antisera against HNF3a, b and g allowed the HNF3 DNA-binding activity to be associated with the transcription factor HNF3 g ( Fig.1B). Treatment with OAT reduces the HNF3 DNA-binding activity within 1 hr after administration and keeps it so low during a months at least (Fig.2A)

Our data suggest that OAT affects the transcription factor HNF3g and therefore, the inhibitory effect, which this hepatocarcinogen has on the glucocorticoid induction of ТАТ, must be the result of reduction of its DNA-binding activity due to initiation by OAT of its direct or indirect modification, which is yet to be found out. As HNF3 is one of the most important liver-enriched transcription factors, responsible for differentiated liver cells phenotype, it suggests the involvement of the prolonged reduction of HNF3 DNA-binding activity in OAT hepatocarcinogenic action.

Acknowledgments

The work was supported by the Russian Foundation for Basic Research (98-04-49434)

 

Table 1. Contents of the GR-TRRD database

 

Functional groups

  

Protein

  

Gene N

  

Regulation

  

Species
I Genes codingfor enzymes Phosphoenolpyruvate(GTP)-carboxykinase G000782 positive

rat
of carbohydrate and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase G000784 positive

rat
amino acids Tyrosine aminotransferase G000798 positive

rat
metabolism Tryptophan oxygenase G000803 positive

rat
Aspartate aminotransferase G001175 positive

rat
II Genes coding Growth hormone G000282 positive

human
for hormones Placental lactogen 2 G000235 positive

human
Prolactin G001206 negative

cow
glycoprotein hormone a -subunit G000271 negative

human
Pro-Opiomelanocortin G000786 negative

rat
Angiotensinogen G000703 positive

rat
Kininogen G000797 positive

rat
III Genes coding Glucocorticoid receptor G000742 negative

rat
for hormone Insulin receptor G000321 positive

human
receptors Angiotensin receptor G001204 positive

rat
IV Viral genes MMTV G000455 positive
and Murine Moloney Sarcoma G001050 positive
genomes HPV-16 G000171 positive
V Genes coding a –acid glycoprotein G000697 positive

rat
for acute a –acid glycoprotein G000463 positive

mouse
phase Haptoglobin G000751 positive

rat
proteins Bb -fibrinogen G001052 positive

frog
Interleukin-6 G000316 negative

human
Metallothionein I G000559 positive

mouse
Metallothionein IIА G000337 positive

human
Gene coding for enzymes Cytochrom CYP2B2 G000729 positive
of xenobiotic metabolism Alcohol dehydrogenese 2 G000185 positive

human
Genes coding for components of antibacterial defence Lysozyme G000064 positive

chiken
Genes coding for b-casein G000709 positive

rat
milk proteins acid whay protein G000629 positive

mouse
Genes a2u-globulin G000700 positive

rat
coding a-fetoprotein G000691 negative

rat
for serum insulin-like growth factor binding protein 1 G000310 positive

human
proteins insulin-like growth factor binding protein 1 G001207 positive

rat
Osteocalcin G000353 negative

human
Osteocalcin G000780 negative

rat
Proliferin G000588 negative

mouse
Link protein G001069 positive

rat
Uteroglobin G000679 positive

rabbit

 

 

Fig. 1. (A) AP1, GME , HNF3 and Ets DNA-binding activity. Every set of data represents: left – the free binding site (at the bottom of the gel), middle – binding site after incubation with nuclear extracts from the livers of intact mice, right – mice treated with the hepatothropic carcinogen OAТ.

(B) Identification of HNF3 isoforms that bind to HNF3 element (A mice). C-control

 

Fig 2.(A) Time course for HNF 3-DNA binding activity in response to OAT at A mice

(B) Changes in HNF 3-DNA binding activity 24 hours after one-time OAT administration

 

References

  1. A.E. Kel., N.A. Kolchanov, O.V. Kel, A.G. Romashchenko, E.A. Ananko, E.V. Ignateva, T.I. Merkulova, O.A. Podkolodnaya, I.L. Stepanenko, A.V. Kochetov, F.A. Kolpakov, N.L. Podkolodnyi, and A.N. Naumochkin, “TRRD: database on transcription regulatory regions of eukaryotic genes” Mol. Biol. 31, 521-530 (1997).
  2. V.I. Kaledin and N.P. Zakharova, In: Investigation on tumor induction and metastasizing in experimental animals. Novosibirsk, 146-185, (1984).
  3. T.I. Merkulova, V.M. Merkulov and R.L. Mitina “Glucocorticoid mechanisms and glucocorticoid controlled gene regulatory regions: Description in the TRRD database” Mol. Biol. 31, 714-726 (1997).
  4. M.S. Miller and G.N. Vogan, “Inhibition of steroid-inducible tyrosine aminotransferase gene expression by N-methyl-N’-nitrosoguanidine in a rat hepatoma cell line”. Carcinogenesis 7, 1273-1278 (1986).
  5. H. Oshima, D. Szapary and S.S. Simons ”The factor binding to the glucocorticoid modulatory element of the tyrosine aminotransferase gene is a novel and ubiquites heteromeric complex”.J.Biol.Chem. 270, 21893-21901(1995).
  6. G.C.T. Yeoh, “The effect of 3’-methyl-4-dimethyl-aminoazobenzene on foetal rat hepatocytes in culture” Eur. J. Cancer Clin. Oncol. 17, 743-752 (1981)