Transcriptional repression of the ectodomain sheddase ADAM10 by TBX2 and potential implication for Alzheimer’s disease
The ADAM10-mediated cleavage of transmembrane proteins regulates cellular processes such as proliferation or migration. Substrate cleavage by ADAM10 has also been implicated in pathological situations such as cancer or Morbus Alzheimer. Therefore, identifying endogenous molecules, which modulate the amount and consequently the activity of ADAM10, might contribute to a deeper understanding of the enzyme’s role in both, physiology and pathology.
To elucidate the underlying cellular mechanism of the TBX2-mediated repression of ADAM10 gene expression, we performed overexpression, RNAi-mediated knockdown and pharmacological inhibition studies in the human neuroblastoma cell line SH-SY5Y. Expression analysis was conducted by e.g. real-time RT-PCR or western blot techniques. To identify the binding region of TBX2 within the ADAM10 promoter, we used luciferase reporter assay on deletion constructs and EMSA/WEMSA experiments. In addition, we analyzed a TBX2 loss-of-function Drosophila model regarding the expression of ADAM10 orthologs by qPCR. Furthermore, we quantified the mRNA level of TBX2 in post-mortem brain tissue of AD patients.
Here, we report TBX2 as a transcriptional repressor of ADAM10 gene expression: both, the DNA-binding domain and the repression domain of TBX2 were necessary to effect transcriptional repression of ADAM10 in neuronal SH-SY5Y cells. This regulatory mechanism required HDAC1 as a co-factor of TBX2. Transcriptional repression was mediated by two functional TBX2 binding sites within the core promoter sequence (− 315 to − 286 bp). Analysis of a TBX2 loss-of-function Drosophila model revealed that kuzbanian and kuzbanian-like, orthologs of ADAM10, were derepressed compared to wild type. Vice versa, analysis of cortical brain samples of AD-patients, which showed reduced ADAM10 mRNA levels, revealed a 2.5-fold elevation of TBX2, while TBX3 and TBX21 levels were not affected.
Our results characterize TBX2 as a repressor of ADAM10 gene expression and suggest that this regulatory interaction is conserved across tissues and species.
KeywordsADAM10 Alpha-secretase APP-processing Kuzbanian Omb TBX2 Transcriptional regulation
A disintegrin and metalloproteinase 10
Amyloid precursor protein
Beta site APP cleaving enzyme-1
Consortium to establish a registry for Alzheimer’s disease
Expressed sequence tag
Electrophoretic mobility shift assay
Histone deacetylase 1
Nerve glia antigen 2
Peroxisome proliferator activated receptor-alpha
Ribosomal protein 49
- SA beta-GAL
Senescence associated beta-galactosidase
Transcripts per million
X-box binding protein-1
This work was supported by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN), FKZ01GS08130, 01GS08125 and 01GS08129-5 and by the Alfons Geib Stiftung. We thank K. Hilger, A. Bruns, and S. Schneider (all University Medical Center Mainz, Germany) for technical assistance; we also are grateful to Inka Hoffmann, Fred Eichinger, and Melanie Heyde (University of Mainz, Germany) for preparation of Drosophila wing imaginal discs and to Sven Grösgen (Saarland University, Germany) for experiments regarding human samples; we want to thank Colin Goding (University of Oxford, UK) for murine TBX2 expression constructs and Christian Haass (LMU Munich, Germany) for APP C-terminal antibody 6687. Stock 25706 obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) was used in this study.
KE conceived and coordinated the study, drafted the manuscript, performed experiments with dermal fibroblasts and generated the TBX2 binding site deletion mutant. SR carried out the molecular biology and biochemistry studies, performed analysis on EST profiles, conducted the statistical analysis, and helped to draft the manuscript. FS constructed secreted luciferase reporter vectors and helped to revise the manuscript. NS performed overexpression experiments of HDAC1 to assess effects on ADAM10 and luciferase reporter gene assays for analyzing the TBX2 binding site deletion mutant. MG and TH designed and coordinated studies regarding human brain tissue and revised the manuscript. GP provided expression constructs for human DDK-tagged TBX2 and Drosophila larval samples. All authors have read and approved the final version of the manuscript.
This work was supported by the German Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN) and FKZ01GS08130 and by the Alfons-Geib Stiftung.
Compliance with ethical standards
Ethical approval and consent to participate
All experiments were conducted in accordance to the official regulations for the care and use of laboratory animals and approved by local authorities (University of Mainz, Germany).
Conflict of interest
The authors declare that they have no competing interests.
- 6.Sakry D, Neitz A, Singh J, Frischknecht R, Marongiu D, Biname F, Perera SS, Endres K, Lutz B, Radyushkin K et al (2014) Oligodendrocyte precursor cells modulate the neuronal network by activity-dependent ectodomain cleavage of glial NG2. PLoS Biol 12:e1001993PubMedPubMedCentralCrossRefGoogle Scholar
- 9.Riddle DL, Blumenthal T, Meyer BJ, Priess JR (1997) Introduction to C. elegans. In: Riddle DL, Blumenthal T, Meyer BJ, Priess JR (eds) C. elegans I. Cold Spring Harbor, New YorkGoogle Scholar
- 12.Hartmann D, de Strooper B, Serneels L, Craessaerts K, Herreman A, Annaert W, Umans L, Lübke T, Lena Illert A, von Figura K, Saftig P (2002) The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts. Hum Mol Genet 11:2615–2624PubMedCrossRefPubMedCentralGoogle Scholar
- 17.McCulloch DR, Akl P, Samaratunga H, Herington AC, Odorico DM (2004) Expression of the disintegrin metalloprotease, ADAM-10, in prostate cancer and its regulation by dihydrotestosterone, insulin-like growth factor I, and epidermal growth factor in the prostate cancer cell model LNCaP. Clin Cancer Res 10:314–323PubMedCrossRefGoogle Scholar
- 26.Milosch N, Tanriover G, Kundu A, Rami A, Francois JC, Baumkotter F, Weyer SW, Samanta A, Jaschke A, Brod F et al (2014) Holo-APP and G-protein-mediated signaling are required for sAPPalpha-induced activation of the Akt survival pathway. Cell Death Dis 5:e1391PubMedPubMedCentralCrossRefGoogle Scholar
- 27.Postina R, Schroeder A, Dewachter I, Bohl J, Schmitt U, Kojro E, Prinzen C, Endres K, Hiemke C, Blessing M et al (2004) A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest 113:1456–1464PubMedPubMedCentralCrossRefGoogle Scholar
- 34.Jacobs JJ, Keblusek P, Robanus-Maandag E, Kristel P, Lingbeek M, Nederlof PM, van Welsem T, van de Vijver MJ, Koh EY, Daley GQ, van Lohuizen M (2000) Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers. Nat Genet 26:291–299PubMedCrossRefGoogle Scholar
- 71.Weber S, Niessen MT, Prox J, Lullmann-Rauch R, Schmitz A, Schwanbeck R, Blobel CP, Jorissen E, de Strooper B, Niessen CM, Saftig P (2011) The disintegrin/metalloproteinase Adam10 is essential for epidermal integrity and Notch-mediated signaling. Development 138:495–505PubMedPubMedCentralCrossRefGoogle Scholar
- 92.Schneider MA, Scheffer KD, Bund T, Boukhallouk F, Lambert C, Cotarelo C, Pflugfelder GO, Florin L, Spoden GA (2013) The transcription factors TBX2 and TBX3 interact with human papillomavirus 16 (HPV16) L2 and repress the long control region of HPVs. J Virol 87:4461–4474PubMedPubMedCentralCrossRefGoogle Scholar
- 93.Habets PE, Moorman AF, Clout DE, van Roon MA, Lingbeek M, van Lohuizen M, Campione M, Christoffels VM (2002) Cooperative action of Tbx2 and Nkx2.5 inhibits ANF expression in the atrioventricular canal: implications for cardiac chamber formation. Genes Dev 16:1234–1246PubMedPubMedCentralCrossRefGoogle Scholar
- 97.Pflugfelder GO, Roth H, Poeck B, Kerscher S, Schwarz H, Jonschker B, Heisenberg M (1992) The lethal(1)optomotor-blind gene of Drosophila melanogaster is a major organizer of optic lobe development: isolation and characterization of the gene. Proc Natl Acad Sci USA 89:1199–1203PubMedCrossRefPubMedCentralGoogle Scholar
- 99.Grimm MO, Grosgen S, Rothhaar TL, Burg VK, Hundsdorfer B, Haupenthal VJ, Friess P, Muller U, Fassbender K, Riemenschneider M et al (2011) Intracellular APP domain regulates serine-palmitoyl-CoA transferase expression and is affected in alzheimer’s disease. Int J Alzheimers Dis 2011:695413PubMedPubMedCentralGoogle Scholar