Before cell motility assay cells were
Before cell motility assay, tepp were pretreated with GW1100 (1 μM), which is an antagonist of GPR40 , . GW1100 increased the cell motile activity of MG63-R7 cells in the presence of GW9508, similar as observed with MG-63 cells (Fig. 4A). Moreover, to confirm the effects of GPR120 on the cell motile activity of MG63-R7 cells, GPR120 knockdown (MG-R7-120) cells were generated from MG63-R7 cells. The cell motile activity of MG-R7-120 cells was significantly lower than that of MG-R7-R (control) cells. While GW9508 elevated the cell motile activity of MG-R7-R cells, the cell motile activity of MG-R7-120 cells was suppressed by GW9508 (Fig. 4B). In addition, MG-R7-120 cells indicated the low invasive activity, compared with MG-R7-R cells (Fig. 4C). In gelatin zymography, the activation of MMP-2 was significantly higher in MG-R7-120 cells than in MG-R7-R cells, while no activation of MMP-9 was detected. GW9508 did not affect MMP-2 activations in both cells (Fig. 4D and E). Taken together, these results showed that GPR120 positively and GPR40 negatively regulated the cell motile activity of highly migratory cells. In contrast, the activity of MMP-2 in highly cell migratory cells was suppressed by GPR120. Our recent studies showed that GPR120 enhanced and GPR40 suppressed the activation of MMP-2 in pancreatic cancer cells, whereas the activation of MMP-2 in lung cancer cells was stimulated by GPR40 , . Therefore, the effects of GPR120 and GPR40 on cellular functions may be dependent on the types of cancer cells.
Conflict of interest statement
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 24590493 and by Grants from the Faculty of Science and Engineering, Kindai University.
Introduction The mammary gland secretes milk for the growth and health of mammalian neonates . The development of mammary gland occurs through various stages, including embryo, puberty, pregnancy, lactation and involution , . It has been demonstrated that inhibition of mammary gland development at puberty results in impaired development and lactation in the subsequent stages , . Thus, avoiding the restriction of pubertal mammary gland development is the prerequisite for the subsequent normal structure and function of mammary gland. The pubertal mammary gland development is influenced by hormones and growth factors , which stimulate the terminal end bud (TEB) formation and ductal branching by inducing epithelial cell proliferation through activation of PI3K/Akt signaling pathway , . Besides hormones and growth factors, diet and nutrition are also involved in the regulation of pubertal mammary gland development , . It has been shown that high fat diet (HFD) leads to stunted mammary duct elongation and reduced mammary epithelial cell proliferation in the mammary gland of pubertal C57BL/6 mice . In addition, our recent study showed that lauric acid, a saturated medium-chain fatty acid (MCFA), exerted promotive effects on the development of mammary gland in pubertal mice . However, the role of saturated long-chain fatty acids (LCFA) in the development of pubertal mammary gland remains to be further elucidated. Stearic acid (SA), a saturated LCFA consisting of 18 carbon atoms, is abundant in the fats such as cocoa butter, mutton tallow, beef tallow, lard and butter . Unlike elevating cholesterol effects of the other saturated LCFAs, SA has been shown to have a neutral effect on blood total and low density lipoprotein cholesterol levels , . Meanwhile, dietary lipid supplements high in SA increased C18:0 concentration in milk fat of lactating dairy cows . In addition, it has been reported that SA alters microRNA profiles in bovine mammary gland epithelial cells . Furthermore, SA exerted inhibitory effects on the proliferation of human lymphocyte ,  and human aortic endothelial cells . However, to our knowledge, there is no reference in the literature to the role of SA in pubertal mammary gland development. It has been well known that free fatty acids elicit key physiological functions via the free fatty acids receptors , . Among them, the free fatty acid receptor 4, also called G protein-coupled receptor 120 (GPR120), which recognizes MC- and LCFAs, has been implicated in modulation of metabolism and energy utilization as well as endocrine and immune function , , , . While whether GPR120 is involved in the regulation of mammary gland development remains unclear.