Dissection of mechanoresponse elements in promoter sites of the mechanoresponsive CYR61 gene

Abstract

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.

NOTCH Signaling Is Activated through Mechanical Strain in Human Bone Marrow-Derived Mesenchymal Stromal Cells

“…BMSCs were obtained from different donors as indicated, and 5 × 10 5 cells per well were seeded on 4-well PU plates, allowed to attach, and cultured for one week. Cells were treated with 2 and 4 μ M GSI XII or the solvent DMSO (0 μ M) for 24 h. PU dishes were placed in a bioreactor, and cyclic stretching was applied as previously described [10, 12]. 15 min and 4 h later, cells were harvested and total RNA was isolated by using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions.…”

“…As a result, mechanoresponsive signaling cascades such as the mitogen activated protein kinase (MAPK) pathway, which translate mechanical signals into the activation of transcription factors such as AP-1 or SP1, are activated. Both factors bind to responsive elements in the nuclei of effector cells [9–12].…”

Interactions between Muscle and Bone—Where Physics Meets Biology

“…Mechanotransduction activates early genes such as FOS, PTGS2 (alias COX2), and also CYR61/CCN1 but also moderately fast gene transcription. Transcription activation is also variably dependent on the amplitude, frequency, and duration of mechanical stimuli [52,54]. Moreover, the sensitivity to mechanical signals in bone is of course strongly modulated by other signaling molecules, as it was for example shown for estrogens and the mechano-related effects on fracture healing [15,17].…”

“…Such mechanoresponsive elements and promoter constructs have been analyzed and characterized by promoter bashing experiments using reporter gene constructs under the control of the iterative deletion of promoter sequences. Using cyclic stretching experiments, we investigated the mechanoresponse of the promoter of the matricellular growth factor cysteine-rich angiogenic inducer 61 (CYR61/CCN1) in human telomerase-immortalized mesenchymal stem cells [52]. Others characterized the response to the compression of a 3 kbp promoter region in the cartilage-related gene cartilage oligomeric matrix protein (COMP) [53].…”

Polymorphisms of the CYR61 gene in patients with acute myeloid leukemia in a Han Chinese population

“…[ 21 ] That intron has been shown to regulate gene expression, [ 22 , 23 ] and it was reported that breast carcinogenesis was accompanied by a shift in intron 3 of the CYR61 gene moving toward a phenotype wherein intron 3 is missing from the mRNA transcript. [ 24 ] It was also demonstrated that the 5′-flanking region of the Cyr61 gene regulated its expression, [ 25 , 26 ] but little is known about the 3′-flanking region of the Cyr61 gene. Although this study provides a clue regarding the roles of CYR61 polymorphisms in AML, whether and how the roles of Cyr61 polymorphisms are associated with hematological tumorigenesis remain unclear and need further study.…”