Mechanical strain induces involution-associated events in mammary epithelial cells

Abstract

Background: Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture.

Results: We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition.

Conclusion: Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device specifically designed for such a purpose. We believe that our results indicate the relevance of mechanical stress among the early post-lactation events that lead to mammary gland involution.

Figure 1

Equibiaxial stretching device. (A) Assembled device and its individual components (1–5): 1) Delrin^® made cylinder with an inner ring that constitutes the silicone membrane holder; 2) Delrin^® made ring with an O-ring that attaches the membrane to the inner holder; 3) Teflon^® made indenter ring; 4) Delrin^® made flange that pushes down piece #3; 5) Aluminum screw-top, that pushes down piece #4. (B) Diagram (in scale) depicting the procedure to assemble the complete device for performing cell stretching protocols; step 1 indicates positioning of the flexible membrane with attached cells grown in a 60 mm Petri dish; following steps indicate assembly of components shown in (A); (C) Cross-section of the complete device. For each piece, specific dimensions are described in millimeters.

Figure 2

Theoretical model to predict membrane strain magnitude. (A) Initial and final position of stretching device (1st row) and flexible membrane (2nd row); equations describing membrane area in each position (3rd row); a: radius of circular stretchable area; b: radius of inner circle where cells can be visualized; h: distance made by the flange (item#4 of Figure 1); c: radius of a hypothetical new circle corresponding to the area of the stretched membrane in the final position (Equation (1)). The relative increment of c/a can be calculated by Equation (2). Equation (3) indicates the percentage of radial strain applied (RSA) to the membrane. (B) The distances between 10 pairs of cells were measured in each orientation (horizontal, vertical and diagonal) in 4 different microscope fields. Data are expressed as means ± SE. Predicted RSA respect to h (from Equation (2) and (3)) is also plot in the graph. (C) Relationship between thread turns and cell area change (%). The areas of 15 cells were measured in four different microcope fields. Data are expressed as means ± SE. The predicted substrate area change (%) is also plot in the graph. (D) Representative image of phase contrast micrographs (magnification 200×) of HC11 cells attached to silicone membranes. Silicone membranes were progressively subjected to 0% (upper panel), 20% (middle panel) and 30% (lower panel) RSA and the same field is shown in each panel. At each RSA, the arrow points out a loosely attached cell, while the arrowhead shows a well attached one. On the left, contours of cells identified on the right are depicted. Relationship between thread-turns and RSA: 1 turn = 0.54 mm.

Figure 3

Effects of mechanical stress on c-Fos expression. mRNA levels were analyzed by quantitative real time RT-PCR in HC11 cells stretched for 1 h using different strain intensities (A) or subjected to 20% RSA for the indicated time periods (B); gapdh expression was used to normalize c-fos mRNA levels. c-Fos protein levels were analyzed by western blot in HC11 cells subjected to 20% RSA for the indicated times (C). Nuclear fractions from HC11 mammary epithelial cells were analyzed by western blot to determine c-Fos protein presence in this compartment after 3 h of sustained 20% RSA (D). Analysis was performed by triplicate in at least three independent experiments. Significant differences: (*) p < 0.05 compared to 0% RSA and (**) p < 0.05 compared to 5% RSA.

Figure 4

Effects of mechanical stress on ERK1/2 and AKT phosphorylation. ERK1/2 (A) and AKT (B) phosphorylation levels were analyzed by western blot in HC11 cells subjected to 20% RSA for the indicated times. Analysis was performed by triplicate in three independent experiments. Data are expressed as means ± SE. Significant differences: (*) p < 0.05 compared with 0 min and (**) p < 0.05 compared with 60 min. Six-hour stretched cells have their own corresponding un-stretched control (grey bar).

Figure 5

Effects of mechanical stress on STAT3 phosphorylation and LIF expression. STAT3 phoshorylation levels were determined by western blot analysis (A) and lif mRNA expression levels were analyzed by quantitative real time RT-PCR (B) in HC11 cells subjected to 20% RSA for the indicated times. In (A) and (B), analysis were performed by triplicate in three independent experiments. Data are expressed as means ± SE. Significant differences: (*) p < 0.05 compared with 0 min and (**) p < 0.05 comparing cells stretched for 6 h with its corresponding control (grey bar). LIF expression levels quantified by ELISA in the conditioned medium (CM) of cells subjected (S) or not (C) to 20% RSA for the indicated times. Results are expressed in ng/ml and represent the mean ± S.E. of three independent experiments. Significant differences: (*) p < 0.05 compared to their corresponding control and to CM of cells stretched for 8 h (C).