Dissection of mechanoresponse elements in promoter sites of the mechanoresponsive CYR61 gene.
Mechanotransduction is important for mesenchymal regeneration and differentiation. Exaggerated high or very low impact yields pathological outcome resulting in fracture or tissue atrophy. Pathological strain in animal models was described but tools to dissect the respective stimuli and downstream pathways are limited. We expand the analytical tools to describe DNA strain response elements in a reporter gene approach. Deletion constructs of the human cysteine-rich protein 61 (CYR61) promoter were cloned into luciferase vectors and stably transfected into human telomerase-immortalised mesenchymal stem cells (hMSC-TERT). Cells were mechanically stimulated with variable frequencies, amplitudes and durations. Promoter activity was determined as well as CYR61 mRNA and protein expression. In silico promoter analysis identified putative transcription factor binding sites, one of which was a cAMP response element, verified by electrophoretic mobility shift assay. We demonstrate for the first time that the activity of promoter regions is inhibited in low, but stimulated in high frequency stimulations. We conclude that by varying conditions of mechanical strain it is possible to characterize stimulatory versus inhibitory strain on cellular levels. Our work may be helpful in future studies to dissect the molecular pathways of physiological versus pathological strain and may have implications for clinical exercise based treatment strategies.
Orthopedic Center for Musculoskeletal Research, Orthopedic Department, University of Würzburg, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany.
Lothar
L
Seefried
Orthopedic Center for Musculoskeletal Research, Orthopedic Department, University of Würzburg, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany.
Sigrid
S
Müller-Deubert
Orthopedic Center for Musculoskeletal Research, Orthopedic Department, University of Würzburg, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany.
Melanie
M
Krug
Orthopedic Center for Musculoskeletal Research, Orthopedic Department, University of Würzburg, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany.
Almoatazbellah
A
Youssef
Orthopedic Center for Musculoskeletal Research, Orthopedic Department, University of Würzburg, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany.
Norbert
N
Schütze
Institute of Orthopedic Research and Biomechanics, Center of Musculoskeletal Research, University of Ulm, Helmholtzstrasse 14, 89081 Ulm, Germany.
Anita
A
Ignatius
Orthopedic Center for Musculoskeletal Research, Orthopedic Department, University of Würzburg, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany.
Franz
F
Jakob
Orthopedic Center for Musculoskeletal Research, Orthopedic Department, University of Würzburg, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany. Electronic address: r-ebert.klh@uni-wuerzburg.de.
Regina
R
Ebert
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003198:Computer Simulation; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D055515:Cysteine-Rich Protein 61 / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013314:Stress, Mechanical; D019098:Telomerase / Q000378:metabolism; D019076:Transgenes
C501848:CCN1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D055515:Cysteine-Rich Protein 61; D012333:RNA, Messenger; D008156:Luciferases; D019098:Telomerase
Mechanotransduction; Promoter analysis; Telomerase-immortalised mesenchymal stem cells; hCyr61