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Mechanical strain induces involution-associated events in mammary epithelial cells.

Quaglino A, Salierno M, Pellegrotti J, Rubinstein N, Kordon EC - BMC Cell Biol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Química Biológica e Instituto de Fisiología, Biología Molecular y Neurociencias-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. quaglino@qb.fcen.uba.ar

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.

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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.
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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.

Mentions: We built a theoretical model that let us to predict the strain exerted on flexible membranes (Figure 2). In this geometrical representation the area outlined by the O-ring was considered the "stretchable surface" and it is shown in the diagram as the circle of radius a ("Initial Position" in Figure 2). In addition, the area defined by the indenter ring is depicted as a circle radius b. When the flange (item #4) is pushed down an h distance, the membrane adopts an "inverted hat" shape of radius a, at the top, and b, at the bottom ("Final Position" in Figure 2). This shape was theoretically re-defined as a new circle of radius c, whose area could be calculated by Equation 1. Then, the relative radial increment of the membrane (c/a) could be calculated by Equation 2 (Figure 2, grey box).


Mechanical strain induces involution-associated events in mammary epithelial cells.

Quaglino A, Salierno M, Pellegrotti J, Rubinstein N, Kordon EC - BMC Cell Biol. (2009)

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.
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Related In: Results  -  Collection

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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.
Mentions: We built a theoretical model that let us to predict the strain exerted on flexible membranes (Figure 2). In this geometrical representation the area outlined by the O-ring was considered the "stretchable surface" and it is shown in the diagram as the circle of radius a ("Initial Position" in Figure 2). In addition, the area defined by the indenter ring is depicted as a circle radius b. When the flange (item #4) is pushed down an h distance, the membrane adopts an "inverted hat" shape of radius a, at the top, and b, at the bottom ("Final Position" in Figure 2). This shape was theoretically re-defined as a new circle of radius c, whose area could be calculated by Equation 1. Then, the relative radial increment of the membrane (c/a) could be calculated by Equation 2 (Figure 2, grey box).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Química Biológica e Instituto de Fisiología, Biología Molecular y Neurociencias-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. quaglino@qb.fcen.uba.ar

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.

Show MeSH
Related in: MedlinePlus