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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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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.
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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.

Mentions: Since c-Fos protein has been implicated in both, cellular response to mechanical stress and mammary gland involution, we proceeded to analyze its expression and activation levels in our experimental setting. Figure 3A shows that c-fos mRNA expression was induced in a dose-dependent manner under rising single-step RSAs (from 0 to 15%) for 1 h. This induction reached a plateau from 15% to 20% RSA and started to decrease at 30% RSA. Then, time course of c-fos mRNA expression in HC11 cells subjected to 20% linear strain was analyzed. We found that the highest expression levels were reached after 30 min of sustained strain (Figure 3B). When c-Fos protein expression levels were determined by western blot analysis, a significant increase was observed after 60 min of 20% RSA (Figure 3C). In addition, under these conditions, nuclear localization of the protein was also verified (Figure 3D) indicating that mechanical stress also induced c-Fos nuclear translocation.


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

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

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2721828&req=5

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.
Mentions: Since c-Fos protein has been implicated in both, cellular response to mechanical stress and mammary gland involution, we proceeded to analyze its expression and activation levels in our experimental setting. Figure 3A shows that c-fos mRNA expression was induced in a dose-dependent manner under rising single-step RSAs (from 0 to 15%) for 1 h. This induction reached a plateau from 15% to 20% RSA and started to decrease at 30% RSA. Then, time course of c-fos mRNA expression in HC11 cells subjected to 20% linear strain was analyzed. We found that the highest expression levels were reached after 30 min of sustained strain (Figure 3B). When c-Fos protein expression levels were determined by western blot analysis, a significant increase was observed after 60 min of 20% RSA (Figure 3C). In addition, under these conditions, nuclear localization of the protein was also verified (Figure 3D) indicating that mechanical stress also induced c-Fos nuclear translocation.

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