Effect of Mechanical Stress in the Mammary Gland Epithelium.

QUAGLINO ANA; TANOSTAMARA; KALINEC FEDERICO; COSO OMAR ADRIÁN; KORDON EDITH CLAUDIA

Biddeford, Maine, EEUU

Congreso; Gordon Reserach Conference in Mechanosensory Transduction; 2007

It has been demonstrated that local factors are responsible for the initiation of mammary gland involution after weaning. Results from different groups, including our own, indicate that during this process there is transcription induction of specific genes, as lif, c-fos and tnf-a, ƒnand activation of different quinases and transcription factors as Erk1/2 and Stat3.  It has been speculated that milk-stasis and the consequent stretching of lobular acini could be the earliest stimulus for triggering this process. However, up to now there has been no report indicating whether mechanical stress could exert any effect on mammary epithelial cells. Therefore, our goal was to determine whether mammary cell stretching, could be an early signal that triggers biochemical changes that in turn would lead to epithelial apoptosis. Then, a novel device that applies controlled radial stretching to cells growing in monolayer was designed for mimicking the physical tension generated in the mammary acini when milk efflux is interrupted. Mammary epithelial HC11 cells were seeded on flexible silicone sheets previously coated with collagen. When cells reached confluency, silicone sheets were stretched up to 20% during different times (2min ¡V 4hs). By semiquantitative and quantitative RT-PCR assays we found that cells exposed to sustained stretching (30 min) showed lif, c-fos and tnf-a transcription induction. In addition, by Western blot analysis we found that mechanical stress also induced Erk1/2 serine and Stat3 tyrosine phosphorylation after 5 minutes. In addition, after 1h a very strong increase in c-Fos protein levels was detected. Interestingly, stretched cells showed a much higher content of this protein in the cell nuclei than controls, suggesting that mechanical stress not only induced c-Fos expression but also its activation. Mechanically stressed cells were explored by confocal microscopy. Analysis of the cytoskeletal organization: actin filaments (F-actin) and microtubules (b-tubulin), showed that prominent stress fibers present in some of the cells dissappeared after 24 hs of sustained stress. b-tubulin expression had a perinuclear distribution compared with controls. These results are the first direct evidence showing that mammary cell stretching could be relevant in triggering events leading to cell death during post-lactational involution