TRPV4 Increases the Expression of Tight Junction Protein-Encoding Genes via XBP1 in Mammary Epithelial Cells

SIMPLE SUMMARY: Mammary glands are exocrine tissue, capable of secreting adequate amounts of milk protein during lactation. Each mammary gland is occupied by numerous alveoli. Each alveolus is composed of a single layer of mammary epithelial cells, adipose tissue, and ducts. Recent studies indicate that mild heat treatment of mammary epithelial cells at 39 °C has activated milk production. These results suggest that temperature may influence the physiological functions of mammary epithelial cells. In this study, we found that the temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) was involved in the increase of β-casein and TJ protein-encoding gene expression in response to mild heat treatment. On the other hand, severe heat treatment (41 °C) reduced the cell viability. Moreover, the Trpv4 mRNA level was significantly increased at Day 15 of gestation when the mammary alveoli are formed. TRPV4 is activated not only by temperature but also by mechanical forces that guide mammary epithelial development in the normal mammary gland. Our data suggest that TRPV4 has a possible function in mammary gland development. ABSTRACT: Mild heat stress (39 °C–40 °C) can positively regulate cell proliferation and differentiation. Indeed, mild heat treatment at 39 °C enhances the less-permeable tight junctions (TJs) formation and milk production in mammary epithelial cells. However, the molecular mechanisms of this response have not yet been delineated. In this study, the involvement of temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) in the increase of β-casein and TJ protein-encoding gene expression in response to mild heat treatment (39 °C) has been explored using HCll mouse mammary epithelial cells. Severe heat treatment (41 °C) induced the transcriptional level of Chop (C/EBP homologous protein; proapoptotic marker) and reduced the cell viability. It is speculated that the difference in unfolded protein response (UPR) gene expression upon stimulation at 39 °C vs. 41 °C controls cell survival vs. cell death. The accumulation of Trpv4 mRNA was significantly higher in 39 °C heat treatment cells. The β-casein, Zo-1 (zona occludens-1), Ocln (occludin), and Cldn3 (claudin 3) transcript levels were significantly increased in response to the addition of a selective TRPV4 channel agonist (GSK1016790A) at 37 °C. TRPV4 stimulation with GSK1016790A also increased the X-box-binding protein 1 splicing form (Xbp1s) at the transcript level. The increase in the mRNA levels of β-casein, Zo-1, Ocln, and Cldn3 in response to 39 °C heat treatment was suppressed by XBP1 knockdown. Moreover, the transcript level of Trpv4 was significantly increased at Day 15 of gestation, and its expression declined after 1 day of lactation. TRPV4 is activated not only by temperature but also by mechanical forces, such as cell stretching and shear stress, which guide mammary epithelial development in a normal mammary gland. These findings provide new insights of the possible function of TRPV4 in mammary gland development.