Regulation of LRRK2 promoter activity and gene expression by Sp1

Abstract

Background: The dopaminergic neurodegeneration in the nigrostriatal pathway is a prominent neuropathological feature of Parkinson's disease (PD). Mutations in various genes have been linked to familial PD, and leucine-rich repeat kinase 2 (LRRK2) gene is one of them. LRRK2 is a large complex protein, belonging to the ROCO family of proteins. Recent studies suggest that the level of LRRK2 protein is one of the contributing factors to PD pathogenesis. However, it remains elusive how LRRK2 is regulated at the transcriptional and translational level.

Results: In this study, we cloned a 1738 bp 5'-flanking region of the human LRRK2 gene. The transcriptional start site (TSS) was located to 135 bp upstream of translational start site and the fragment -118 to +133 bp had the minimum promoter activity required for transcription. There were two functional Sp1- responsive elements on the human LRRK2 gene promoter revealed by electrophoretic mobility shift assay (EMSA). Sp1 overexpression promoted LRRK2 transcription and translation in the cellular model. On the contrary, application of mithramycin A inhibited LRRK2 transcriptional and translational activities.

Conclusion: This is the first study indicating that Sp1 signaling plays an important role in the regulation of human LRRK2 gene expression. It suggests that controlling LRRK2 level by manipulating Sp1 signaling may be beneficial to attenuate PD-related neuropathology.

Keywords: Gene regulation; LRRK2; Parkinson’s disease; Sp1.

Fig. 1

Identification of TSS and sequence features of the human LRRK2 gene promoter. a Smarter RACE cDNA amplification kit was used to amplify full-length cDNA from HEK293 cells. Nested PCR was performed and the product was resolved on 1.5 % agarose gel. b TSS was located by sequencing PCR product. The first base pair after SMARTer oligonucleotide is the TSS, which is indicated by arrow in Figure. c Sequence of the human LRRK2 promoter from -1865 bp to + 213 bp of the TSS (+1) is illustrated here. The putative transcription factor binding sites are underlined by computational search

Fig. 2

Functional deletion analyses of the human LRRK2 gene promoter. a Schematic illustration of human LRRK2 promoter constructs consisting a serial deletion fragments, which were cloned into pGL3-Basic plasmid. The arrows represent the direction of transcription and the numbers indicate the start and ending point of each construct with respect to TSS. b LRRK2 promoter constructs were verified by restriction enzyme digestion and the digested products were resolved on 1.5 % agarose gel. The size of vector is 4.8 kb and the size of inserts ranges from 84 to 1871 bp, which was further confirmed by sequencing. c Plasmids with different LRRK2 promoter constructs were cotransfected with pCMV-Luc into HEK293 cells. Cell lysates were harvested 24 h post-transfection, and the luciferase activity of pCMV-luc was used for normalizing transfection efficiency. The RLU of pGL3-Basic (marked as N) was designated as 1. The values represent means ± SEM, n =3, *p < 0.001, by analysis of variance (ANOVA) with Sidak’s multiple comparison test. Comparisons were made between all other columns and the pGL3-basic control column

Fig. 3

Regulation of the human LRRK2 gene promoter by Sp1. a pGL3-Basic, pLRRK2-C and pLRRK2-J plasmids were cotransfected with either vector or Sp1 expression plasmid into HEK293 cells. Cell harvesting and the measurement of luciferase activities were performed as mentioned before. Sp1 overexpression significantly increased the promoter activity of pLRRK2-C but had no effect on pLRRK2-J nor pGL3-basic control. The values represent means ± SEM. n =3, *p < 0.01 by two way ANOVA with Sidak’s multiple comparison test. b pGL3-Basic, pLRRK2-C and pLRRK2-J plasmids were cotransfected with either negative control or Sp1 siRNA into HEK293 cells. Knockdown of Sp1 significantly decreased the promoter activity of pLRRK2-C but had no effect on pLRRK2-J. The values represent means ± SEM. n =3, *p < 0.01 by two-way ANOVA with Sidak’s multiple comparison test. c EMSA was performed as described in detail in Material and Methods. Sp1 consensus binding sequence was labelled by fluorescent IR700 Dye. Lane1 is the labelled probe alone without nuclear protein extract. Incubation the probes with Sp1-enriched nuclear protein extracts formed a shifted DNA-protein complex band (lane 2). Competition assays were conducted by adding various concentrations of molar excess of unlabeled competitive oligonucleotides, including consensus Sp1 oligonucleotides (lane 3 and 4), mutant Sp1 consensus oligonucleotides (lane 5 and 6) and putative Sp1-responsive elements in the human LRRK2 promoter (lane 7 to 12)

Fig. 4

Sp1 upregulates the LRRK2 gene expression. a-d Sp1 overexpression increased LRRK2 mRNA expression level. Sp1 expression plasmid was transfected into HEK293 cells (a) or MN9D cells (c). Cell lysates were harvested 48 h after transfection and total RNA was isolated for RT-PCR. The products of amplified LRRK2 and β-actin genes were analyzed on a 1.5 % agarose gel. Quantification was performed by ImageJ software and endogenous LRRK2 mRNA level was normalized against β-actin. e HEK292 cells were transfected with scrambled siRNA or Sp1 siRNA, and endogenous LRRK2 mRNA level was measured by RT-PCR after 48 h and analyzed on a 1.5 % agarose gel. g HEK293 cells were transfected as mentioned before and cell lysates were harvested 48 h after transfection. Endogenous LRRK2 protein and overexpressed Sp1 were examined by immunoblotting. i HEK293 cells were transfected with negative control siRNA or Sp1 siRNA. After 48 h, cell lysate was harvested for determining LRRK2 and Sp1 protein level by immunoblotting. Quantification of the band intensity in (f), (h), and (j) was performed by ImageJ software. The values in this figure represent means ± SEM. n =3, *p < 0.05, analyzed by Student’s t-test

Fig. 5

MTM inhibits the LRRK2 gene expression. a pGL3-Basic, pLRRK2-C or pLRRK2-J was transfected into HEK293 cells. After 24 h, transfected cells were treated with MTM at 125 nM or vehicle for 24 or 48 h. Luciferase activities were determined as mentioned before, and pCMV-Luc luciferase activity was used for transfection efficiency normalization. b HEK293 cells were transfected with pGL3-Basic, pLRRK2-C or pLRRK2-J. The next day, cells were exposed to MTM at 25, 75 and 125 nM for 24 h. Luciferase activities were measured. The values in (a) and (b) represent the mean ± SEM. n = 3, *p < 0.001 by two-way ANOVA with Sidak’s multiple comparison test. c HEK293 cells were treated with 125 nM MTM or vehicle for 24 h. The LRRK mRNA levels were determined by RT-PCR and normalized against the levels of β-actin. d Quantification of LRRK2 and β-actin mRNA levels in HEK293 cell were completed by ImageJ software. e Cell lysates harvested from HEK293 cells treated with 125 nM MTM or vehicle for 24 h were analyzed by immunoblotting with anti-LRRK2 antibody. β-actin was used as the internal control for protein loading. f Quantification of LRRK2 and β-actin protein levels in HEK293 cell was completed by ImageJ software. g Dopaminergic MN9D cells were treated with 125 nM MTM or vehicle for 24 h. The LRRK mRNA levels were determined by RT-PCR and normalized against the levels of β-actin. h LRRK2 and β-actin mRNA level in MN9D cell was quantified by ImageJ software. The values in (d), (f) and (h) represent the mean ± SEM. n = 3, *p < 0.001 by Student’s t-test