The mammary gland epithelium undergoes dramatic changes during the successive stages of pregnancy, lactation and involution. In the fully lactating gland, the layer of ductal and alveolar mammary cells form coherent sheets in which cell junctions provide a compact permeability barrier. It has been indicated that mechanical stress imposed by milk secretion on these tightly attached cells after parturition is one of the main factors that determine initiation of the ejection reflex [18
]. On the other hand, soon after weaning, when those epithelial junctions are still intact, milk accumulation might initiate involution-associated events.
In an effort to understand mechanical strain contribution to mammary gland involution, we have designed, built and validated a new cell-stretching device. The apparatus presented in this study is inspired in the system previously described by Lee and coworkers [12
], but it allows a wider RSA range. Here we show that this device is useful to apply dose-dependent, homogeneous equibiaxial strain to mammary epithelial cells growing on deformable silicone membranes. Further, mechanical strain transmission to the cultured cells was confirmed by cell area changes shown in Figure and . These results show, as it was previously demonstrated in other models [12
], that the strain applied to the collagen-coated silicone membranes is very similar to the one exerted to the cells cultured on these membranes. Interestingly, we found relatively larger errors in the percentage of cell area change with RSAs higher than 20% (4 thread turns) (see Figure ). Microscopic observations suggested that these relatively larger statistic errors might be due to variations among cells in the ability to adapt to high strain intensities. Excessive cell stretching seemed to cause changes in the attachment of various cells, which could lead to a lower strain transmission from the substrate to these cells (so their area did not increase at the same rate) and/or to an uneven strain distribution within them (causing changes in cell shape).
Different devices that use elastic membranes substrates to support cell adhesion and transmit strain have been previously described [12
]. Some of them, like the one made commercially available by Flexcell International, depend on a computer controlled vacuum unit to exert cyclic or static strain to flexible membranes [23
]. However, even providing a very precise stimulus control, these devices are complex and often require technical and/or electronic assistance, what leads to a significant cost increment. Therefore, we believed that there was a need for new practical, flexible and affordable devices to easily analyze the biological responses of cultured cells to mechanical stress. In fact, not only our device has been developed as a result of this necessity, there were others very recently introduced, as the one presented last year by Rhana et al
]. In this apparatus, flange displacement was also used to generate membrane deformation, but the design was made for 6-well plates instead of single 60 mm dishes.
The device described herein shows the following properties: 1) easy real time optical monitoring of cell geometry, function, and deformation upon strain application; 2) a small size that allows long-term stretching protocols to be performed in a cell incubator; 3) easy sterilization by autoclaving and 4) low cost, which permits the construction of multiple units that can be used to test simultaneously different experimental conditions (e.g. strain levels, intervals of static stress, pharmacological treatments, etc.).
The intensity of mechanical strain imposed on mammary epithelium by milk accumulation after weaning is not known. However, in most experiments reported herein, a 20% RSA was applied. We have chosen that stretching strength because it did not cause any cell damage and was sufficient to activate various signaling pathways in different cell types [26
]. Besides, other studies carried out in epithelial cells have been done using 20% RSA [31
]. In our laboratory, observations in vivo
are underway trying to establish the strain range experienced by the alveolar cells upon weaning. We hope this analysis will help us to determine the biological threshold for triggering mammary involution associated events.
Once the device was developed, we first analyzed the effect of mechanical strain on c-Fos expression in the HC11 mammary epithelial cells. This protein interacts with the Jun family members generating AP-1 transcription factors that bind to DNA in specific regions [32
]. AP-1 plays a very important role in controlling expression of different genes that have a great impact on cell fate decisions [33
]. In addition, this factor has been associated with stress-induced apoptosis in several cell types [38
]. Marti and colleagues have reported that AP-1 expression and activation is linked to apoptosis induction during the early phase of the mouse mammary gland involution. They observed that c-fos
mRNA species were induced in the mammary epithelium soon after weaning, suggesting that AP-1 may be a nuclear regulator of post-lactational involution [39
]. Interestingly, it has also been reported that c-Fos expression is up-regulated by mechanical stress, in vitro
and in vivo
, participating in different specific cellular responses in cardiomyocytes [40
] osteoblasts [42
]and pulmonary epithelial cells [43
]. Our results clearly indicate that mechanical strain is capable of inducing c-Fos transcription and nuclear translocation in mammary epithelial cells, suggesting that they might be very early events during the involution process.
Three Mitogen-activated protein kinase (MAPK) families have been well characterized: the extracellular-regulated protein kinase (ERK1/2), the c-Jun NH2-terminal protein kinase (JNK), and the p38 (the last two are also known as stress-activated protein kinases or SAPKs). Upon activation through tyrosine and threonine phosphorylation, these proteins translocate to the nucleus and phosphorylate transcription factors, such as AP-1 family members and the serum response factor (SRF) [44
]. MAPK activation, have been implicated in mechanically induced signalling in various cell types. For example, in smooth muscle cells, ERK1/2 has been reported to be involved in mechanical stress-induced c-Fos expression [16
]. Interestingly, ERK1/2 activation was also detected during the early phase of mammary gland involution in vivo
]. Our results indicate that ERK1/2 phosphorylation is rapidly increased in mammary epithelial cells subjected to the same mechanical strain that generated c-Fos induction. Therefore, it is possible that ERK1/2 activation induced by mechanical strain may be modulating c-Fos expression induction and activation as previously observed in other models [16
Not only ERK1/2, but also STAT3 might be implicated in c-Fos expression induction and activation in epithelial cells [47
]. In the case of c-fos
gene transcription, it has been determined that both STAT3 and the ERK-mediated pathway co-operate in its induction. In fact, Kunisada et al
] showed that dominant-negative STAT3 or a MEK inhibitor, PD98059, inhibit LIF-induced c-fos
mRNA expression in cardiac myocites. Therefore, the same signaling pathways might be interacting in mechanically stressed mammary epithelial cells.
One of the most critical molecular changes associated with apoptosis induction during mammary gland involution is STAT3 activation via the Janus kinase (JAK) pathway in response to cytokines and growth factors [17
]. It has been demonstrated that shortly after weaning, LIF expression is induced in the mammary epithelium [8
] and it has been reported that this cytokine is the main responsible for STAT3 activation in mammary epithelial cells [8
]. Interestingly, it has been shown that LIF expression is induced by hemodynamic overload in the adult mammalian heart [50
] and our results show that cell stretching induced STAT3 phosphorylation after 15 min of sustained strain. However, LIF secreted by the stretched HC11 cells was only detected in the conditioned medium (CM) after several hours of sustained strain (see Figure ). Therefore, we believe this STAT3 early activation would not be due to an endocrine/paracrine LIF action. Alternatively, it is possible that SRC kinase might be involved in p-STAT3 induction in the HC11 cells as it has been previously found in mechanically induced pulmonary epithelial cells [51
] and smooth muscle cells [52
Levels of p-STAT3 significantly decreased after 1 h and were recovered after 6 h (Figure ). We believe that the second wave of STAT3 activation might be due to LIF secreted by the stretched cells (see Figure ). In fact, preliminary data from our laboratory indicates that CM collected from stretched cells (6–24 h of 20% sustained strain) induced STAT3 phosphorylation in non-stretched HC11 cells. Noteworthy, this effect was inhibited when CMs were pre-incubated with a LIF blocking antibody.
Several studies regarding the impact of mechanical stimuli on protein kinase B/AKT (PKB/AKT) activation have been described in endothelial cells [53
], in vascular smooth muscle [54
] and keratinocytes [55
]. In this study, we observed that mechanical strain triggered p-AKT transient down-regulation in mammary epithelial cells (see Figure ). In the mammary gland, the relevance of shutting down the PI(3)K/AKT pathway after weaning has been demonstrated when activated-AKT transgenic mice showed significant delay in the involution process [56
]. In addition, it has been reported that expression of the PI(3)K negative regulatory subunits (p55alpha and p50alpha), which inhibited AKT phosphorylation, were induced by STAT3 during mammary regression [58
]. These studies indicate that AKT activation may provide a critical cell survival signal that has to be turned-down during mammary involution. Therefore, we believe that stretching induced p-AKT down-regulation might sensitize epithelial cells to undergo apoptosis. However, more experiments need to be done to determine whether or not STAT3 activation mediates this effect in our model.
In spite of p-AKT down-regulation, we have not detected apoptosis induction (analyzed by caspase-3 activation) in the stretched HC11 cells. This observation has different possible explanations. First, it has been reported that in smooth muscle cells, mechanical stress induced apoptosis is p53-dependent [59
]. Therefore, the lack of apoptosis induction in the stretched HC11 cells might be solely due to the lack of wild type p53 expression in this cell line [60
]. Second, it has been reported that in vivo
active (cleaved) caspase-3 was observed only in the shed cells at 12 h and 24 h involution and not in the alveolar wall until 72 h [61
]. Therefore, during mammary involution, although the apoptosis program is initiated before cells detach, the final events would not occur until these cells are removed from the epithelial layer. In this scenario, it is conceivable that an early involution-associated event as mechanical stress may prepare the epithelial cells to die, but would not be enough to trigger the whole apoptotic program.
It is important to point out that the experiments showed herein were performed in confluent, but not differentiated, HC11 cells. We observed that grown on silicon as a substrate, these cells show similar features to competent HC11 cells grown on plastic. We observed high expression of STAT5A and low levels of p-STAT5 and β-casein compared to cells treated with lactogenic hormones. Expression levels of these proteins did not significantly change upon stretching (data not shown). However, we do not know whether mechanical stress would be able to block the action of lactogenic hormones and/or would be able to trigger cell death in fully differentiated cells. More experiments are being performed to answer these questions.
We have previously reported that tumor cell secreted LIF was able to decrease HC11 cell viability [15
]. Here, we show that cells stretched for up to 24 h did not undergo apoptosis, but were able to secrete up to 0.8 ng/ml of LIF (Figure ). New experiments carried out in our laboratory have shown that LIF secreted by mechanically stressed cells was able to induce STAT3 phosphorylation in non-stretched cell (data not shown). Therefore, luminal cells bearing mechanical stress might not be the first to die, but could initiate a domino effect that may lead to massive apoptosis in the mammary epithelium.