Autophagy regulates functional differentiation of mammary epithelial cells

Mitochondria operate as a central hub for many metabolic processes by sensing and responding to the cellular environment. Developmental cues from the environment have been implicated in selective autophagy, or mitophagy, of mitochondria during cell differentiation and tissue development. Mitophagy occurring in this context, termed programmed mitophagy, responds to cell state rather than mitochondrial damage and is often accompanied by a metabolic transition. However, little is known about the mechanisms that engage and execute mitophagy under physiological or developmental conditions. As the mammary gland undergoes post-natal development and lactation challenges mitochondrial homeostasis, we investigated the contribution of mitochondria to differentiation of mammary epithelial cells (MECs). Using lactogenic differentiation of the HC11 mouse MEC line, we demonstrated that HC11 cells transition to a highly energetic state during differentiation by engaging both oxidative phosphorylation and glycolysis. Interestingly, this transition was lost when autophagy was inhibited with bafilomycin A₁ or knockdown of <i>Atg7</i> (<i>autophagy related 7</i>). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover <i>in vitro</i> with the fluorescent probe, <i>pMitoTimer</i>. Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of <i>Prkn/parkin</i> in HC11 cells. We found that MEC differentiation was impaired in <i>shPrkn</i> cells, implying that PRKN is required for MEC differentiation. These studies suggest a novel regulation of MEC differentiation through programmed mitophagy and provide a foundation for future studies of development and disease associated with mitochondrial function in the mammary gland. <b>Abbreviations</b>: AA: antimycin A; ATG5: autophagy related 5; BAF: bafilomycin A₁; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; COX8A: cytochrome c oxidase subunit 8A; CQ: chloroquine; CSN2: casein beta; ECAR: extracellular acidification rate; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone; FUNDC1: FUN14 domain containing 1; HIF1A: hypoxia inducible factor 1 subunit alpha; L1: lactation day 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEC: mammary epithelial cell; mitoQ: mitoquinol; mROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; P: priming; P16: pregnancy day 16; PARP1: poly(ADP-ribose) polymerase 1; PINK1: PTEN induced kinase 1; PPARGC1A: PPARG coactivator 1 alpha; PRKN: parkin RBR E3 ubiquitin protein ligase; <i>shNT</i>: short hairpin non-targeting control; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; TEM: transmission electron microscopy; TFAM: transcription factor A, mitochondrial; U: undifferentiated.

线粒体是众多代谢过程的核心枢纽，通过感知并响应细胞环境发挥功能。环境中的发育信号已被证实与细胞分化及组织发育过程中发生的线粒体选择性自噬（mitophagy）相关。在此情境下发生的线粒体自噬被称为程序性线粒体自噬，其响应的是细胞状态而非线粒体损伤，且通常伴随代谢转换。然而，目前对于生理或发育条件下启动并执行线粒体自噬的分子机制仍知之甚少。
由于乳腺在产后发育及泌乳过程中会挑战线粒体稳态，我们探究了线粒体对乳腺上皮细胞（mammary epithelial cell, MEC）分化的调控作用。利用HC11小鼠乳腺上皮细胞系的泌乳分化模型，我们证实HC11细胞在分化过程中通过激活氧化磷酸化与糖酵解双通路，转变为高能量代谢状态。有趣的是，当使用巴弗洛霉素A₁（bafilomycin A₁）抑制自噬或敲低<i>Atg7</i>（autophagy related 7）基因后，该代谢转换过程消失。
为评估线粒体的特异性靶向情况，我们借助荧光探针pMitoTimer在体外（in vitro）追踪线粒体的氧化状态与更新速率。实验结果确证，细胞分化过程中激活了线粒体自噬。为进一步探究线粒体自噬在分化过程中的必要性，我们在HC11细胞中敲低了<i>Prkn/parkin</i>基因的表达。结果显示，shPrkn细胞的乳腺上皮细胞分化过程受到损伤，表明PRKN对于乳腺上皮细胞分化是必需的。
本研究揭示了一条通过程序性线粒体自噬调控乳腺上皮细胞分化的全新途径，为后续研究乳腺线粒体功能相关的发育与疾病进程奠定了基础。**缩写说明**：AA：抗霉素A（antimycin A）；ATG5：自噬相关5（autophagy related 5）；BAF：巴弗洛霉素A₁（bafilomycin A₁）；BNIP3：BCL2相互作用蛋白3（BCL2 interacting protein 3）；BNIP3L/NIX：BCL2相互作用蛋白3样（BCL2 interacting protein 3 like）；COX8A：细胞色素c氧化酶亚基8A（cytochrome c oxidase subunit 8A）；CQ：氯喹（chloroquine）；CSN2：β-酪蛋白（casein beta）；ECAR：细胞外酸化率（extracellular acidification rate）；FCCP：三氟甲氧基羰基氰对苯腙（trifluoromethoxy carbonylcyanide phenylhydrazone）；FUNDC1：FUN14结构域包含蛋白1（FUN14 domain containing 1）；HIF1A：缺氧诱导因子1亚基α（hypoxia inducible factor 1 subunit alpha）；L1：泌乳第1天（lactation day 1）；MAP1LC3B：微管相关蛋白1轻链3β（microtubule associated protein 1 light chain 3 beta）；MEC：乳腺上皮细胞（mammary epithelial cell）；mitoQ：线粒体泛醇（mitoquinol）；mROS：线粒体活性氧（mitochondrial reactive oxygen species）；OCR：耗氧率（oxygen consumption rate）；P：预激活；P16：妊娠第16天（pregnancy day 16）；PARP1：多聚ADP-核糖聚合酶1（poly(ADP-ribose) polymerase 1）；PINK1：PTEN诱导激酶1（PTEN induced kinase 1）；PPARGC1A：PPARG辅激活因子1α（PPARG coactivator 1 alpha）；PRKN：parkin RBR型E3泛素蛋白连接酶（parkin RBR E3 ubiquitin protein ligase）；shNT：非靶向短发RNA对照（short hairpin non-targeting control）；SQSTM1：自噬受体SQSTM1（sequestosome 1）；STAT3：信号转导与转录激活因子3（signal transducer and activator of transcription 3）；TEM：透射电子显微镜（transmission electron microscopy）；TFAM：线粒体转录因子A（transcription factor A, mitochondrial）；U：未分化（undifferentiated）。