Haem repression of the housekeeping 5-aminolaevulinic acid synthase gene in the hepatoma cell line LMH

Abstract

Haem is essential for the health and function of nearly all cells. 5-Aminolaevulinic acid synthase-1 (ALAS-1) catalyses the first and rate-controlling step of haem biosynthesis. ALAS-1 is repressed by haem and is induced strongly by lipophilic drugs that also induce CYP (cytochrome P450) proteins. We investigated the effects on the avian ALAS-1 gene promoter of a phenobarbital-like chemical, Glut (glutethimide), and a haem synthesis inhibitor, DHA (4,6-dioxoheptanoic acid), using a reporter gene assay in transiently transfected LMH (Leghorn male hepatoma) hepatoma cells. A 9.1 kb cALAS-1 (chicken ALAS-1) promoter-luciferase-reporter construct, was poorly induced by Glut and not by DHA alone, but was synergistically induced by the combination. In contrast, a 3.5 kb promoter ALAS-1 construct was induced by Glut alone, without any further effect of DHA. In addition, exogenous haem (20 microM) repressed the basal and Glut- and DHA-induced activity of luciferase reporter constructs containing 9.1 and 6.3 kb of ALAS-1 5'-flanking region but not the construct containing the first 3.5 kb of promoter sequence. This effect of haem was subsequently shown to be dependent on the -6.3 to -3.5 kb region of the 5'-flanking region of cALAS-1 and requires the native orientation of the region. Two deletion constructs of this approx. 2.8 kb haem-repressive region (1.7 and 1.1 kb constructs) retained haem-dependent repression of basal and drug inductions, suggesting that more than one cis-acting elements are responsible for this haem-dependent repression of ALAS-1. These results demonstrate that there are regulatory regions in the 5'-flanking region of the cALAS-1 gene that respond to haem and provide a basis for further investigations of the molecular mechanisms by which haem down-regulates expression of the ALAS-1 gene.

Figure 1. Partial restriction map of the 5′-UTR and 5′-flanking region of the cALAS-1 gene, and structures of cALAS-1 constructs

Numbering starts at the transcription start point (+1). Recognition sites for restriction endonucleases XhoI, PacI, SmaI and HindIII used for plasmid construction are shown. The portions of the 5′-flanking region and 5′-UTR cloned into the reporter (Luc) gene plasmids (pGL3-Basic or pGL3-Promoter) are indicated, as are the abbreviations used to denote these constructs. A shorter construct, composed of the first 299 bp upstream of the transcription starting point, linked to the luciferase gene, was also studied. It showed no effect of up- or down-regulation.

Figure 2. Effects of Glut and DHA on pGcALAS3.5-Luc (A) or pGcALAS9.1-Luc (B) transiently transfected into LMH cells

Following transfection, LMH cells were treated with vehicle (Me₂SO), Glut (50 μM), or DHA (250 μM), alone or in combination, for 16 h prior to harvest. Luciferase and β-gal activities and protein concentrations were assayed as described in the Materials and methods section. Luciferase activities are expressed as fold induction compared with vehicle-treated control, normalized to β-gal activity and protein concentrations. Results are means±S.E.M. for triplicate determinations, and representative of six independent experiments. *Differs from Me₂SO or DHA alone, P<0.01. In (A), the two groups treated with Glut did not differ significantly from one another. **Differs from all others, P<0.001.

Figure 3. Effect of exogenous haem on pGcALAS9.1-Luc activity

LMH cells were transiently transfected with pGcALAS9.1-Luc and treated with increasing concentrations of haem (‘Heme’; 0, 5, 10, 15, 20 and 40 μM) dissolved in Me₂SO. After 16 h, the cells were harvested and lysates were used to measure luciferase, β-gal and protein levels, as described in the Materials and methods section. Results are means±S.E.M. for triplicate determinations, and are representative of four independent experiments. Luciferase activities are expressed as fold induction compared with vehicle-treated control, normalized to β-gal and protein levels. The amount of Me₂SO added per well was kept constant and was not greater than 2 μl/ml medium, a volume that was shown in previous studies to not affect luciferase activity [26,28,45,46]. *Differs from cells transfected with pGcALAS9.1-Luc and treated with 0 μM haem, P<0.001.

Figure 4. Effects of Glut, DHA and haem in LMH cells transiently transfected with pGcALAS3.5-Luc (A), pGcALAS9.1-Luc (B) or pGcALAS6.3-Luc (C)

Transfected cells were harvested 16 h after chemical treatment, and lysates were used to measure luciferase, β-gal and protein levels. Concentrations of the chemicals were as follows: Glut, 50 μM; DHA, 250 μM; and haem, 20 μM. Results are means±S.E.M. for triplicate determinations, and representative of five independent experiments. Luciferase activities are expressed as fold induction compared with vehicle-treated control, normalized to β-gal and protein levels. *Differs from Glut+DHA, P<0.001. **Differs from others, P<0.001. ¶Differs from Me₂SO, haem or DHA alone, P<0.01. In (A), the three groups treated with Glut did not differ significantly from one another. †Significant decrease from cells treated with Me₂SO, P<0.001.

Figure 5. Effects of Glut and haem on LMH cells transiently transfected with pGL3DRESLuc (A), pGcALASDRES6.3-3.5-Luc (B) or pGcALASDRES3.5-6.3-Luc (C)

Transfected cells were treated, harvested and assayed as described in the Materials and methods section and the legend to Figure 4. Results are means±S.E.M. for triplicate determinations and representative of five independent experiments. **Glut differs significantly from vehicle (Me₂SO) alone or haem alone, P<0.001; *Glut + Haem differs significantly from Glut alone, P<0.001. †Significant decrease from cells treated with Me₂SO, P<0.01.

Figure 6. Effects of Glut, DHA and haem on LMH cells transiently transfected with pGcDRES 6.3-4.5-Luc (A) or pGL3 DRES 4.5-3.5-Luc (B)

Transfected cells were treated, harvested and assayed as described in the Materials and methods section and the legend to Figure 4. Results are means±S.E.M. for triplicate determinations and representative of five independent experiments. †Differs from vehicle alone (Me₂SO) or Glut + Haem, P<0.01. **Differs from all other treatment groups, P<0.0001. *Differs from Glut alone, P<0.001.

Figure 7. Effects of the regulatory haem pool on hepatic haem metabolism

Haem exerts regulatory effects on both ALAS-1 and HO-1 to control both its synthesis and degradation. ALAS-1 catalyses the formation of ALA from glycine and succinyl-CoA. Haem is synthesized from ALA through a series of intermediary steps catalysed by the enzymes of the haem biosynthetic pathway. Several steps in the production of functional mature ALAS-1 protein are down-regulated by exogenous or endogenous haem, as indicated by the minus signs, or by the plus sign indicating increased degradation of ALAS-1 mRNA. Haem is also incorporated into apo-haemoproteins [such as CYP (cytochrome P450) proteins and mitochondrial cytochromes] as the prosthetic group to form functional holo-haemoproteins.