Intracellular Calcium links Milk Stasis to Lysosome Dependent Cell Death during Early Mammary Gland Involution

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning results in the distension of the alveolar structures by the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 hours of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GcAMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression and prior to significant activation of LIF, IL6 and TGFb3 production or LDCD. Interestingly, we observed the activation of TFEB led by activation of TGFb signaling and deactivation of mTOR signaling in the involution of mammary gland. Finally, we demonstrate that increased intracellular calcium activates STAT3 by degrading SOCS3 mediated by TGFb signaling. In summary, these data suggest that intracellular calcium serves as an important biochemical signal linking milk stasis to STAT3 activation, increased lysosome biogenesis, and lysosome-mediated cell death. Overall design: mRNA profile in control, high calcium (10mM calcium with ionomycin) and TGFb3 treated MCF10A cells

乳腺泌乳后的退化（mammary gland involution）是协调性细胞死亡的典型范例。断奶会因乳汁蓄积引发肺泡结构扩张，进而激活信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3），并启动半胱天冬酶非依赖性但溶酶体依赖性细胞死亡（lysosome-dependent cell death, LDCD）通路。尽管STAT3与LDCD在乳腺早期退化中的重要性已得到充分证实，但乳汁淤滞如何激活STAT3的机制仍未完全阐明。
本研究证实，在实验性乳汁淤滞发生后的2~4小时内，质膜钙ATP酶2（PMCA2）钙泵的蛋白表达水平显著下调。PMCA2表达下调与体内胞质钙水平升高相关，该结果可通过GcAMP6f荧光的多光子活体成像进行检测。上述事件与核磷酸化STAT3（pSTAT3）表达的出现同步发生，且早于白血病抑制因子（leukemia inhibitory factor, LIF）、白细胞介素6（interleukin 6, IL6）及转化生长因子β3（transforming growth factor β3, TGFβ3）的大量合成激活或LDCD的启动。
有趣的是，本研究在乳腺退化过程中观察到：转化生长因子β（TGFβ）信号通路的激活可介导转录因子EB（transcription factor EB, TFEB）的活化，同时伴随哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）信号通路的失活。最后，本研究证实，胞内钙水平升高可通过TGFβ信号通路介导的细胞因子信号转导抑制因子3（suppressor of cytokine signaling 3, SOCS3）降解激活STAT3。
综上，本研究数据表明，胞内钙作为关键生化信号，将乳汁淤滞与STAT3激活、溶酶体生物发生增强及溶酶体介导的细胞死亡关联起来。
实验设计：对对照组、高钙组（含10mM氯化钙与离子霉素）以及经TGFβ3处理的MCF10A细胞进行mRNA表达谱分析。