ABSTRACT

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 Atg7 (autophagy related 7). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover in vitro with the fluorescent probe, pMitoTimer. Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of Prkn/parkin in HC11 cells. We found that MEC differentiation was impaired in shPrkn 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.

Abbreviations: 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; shNT: 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.

摘要

线粒体通过感知和响应细胞环境，作为许多代谢过程的中心枢纽。来自环境的发育线索与细胞分化和组织发育过程中线粒体的选择性自噬或线粒体自噬有关。在这种情况下发生的线粒体自噬，称为程序性线粒体自噬，对细胞状态而非线粒体损伤做出反应，并且通常伴随着代谢转变。然而，关于在生理或发育条件下参与和执行线粒体自噬的机制知之甚少。由于乳腺经历出生后发育和哺乳挑战线粒体稳态，我们研究了线粒体对乳腺上皮细胞 (MEC) 分化的贡献。使用 HC11 小鼠 MEC 系的催乳分化，我们证明了 HC11 细胞在分化过程中通过参与氧化磷酸化和糖酵解转变为高能量状态。有趣的是，当巴弗洛霉素 A 抑制自噬时，这种转变就消失了₁或Atg7 的敲低（自噬相关 7）。为了评估线粒体的特异性靶向，我们使用荧光探针pMitoTimer在体外追踪线粒体氧化和周转。事实上，我们发现分化涉及线粒体自噬。为了进一步评估分化过程中线粒体自噬的需求，我们敲低了HC11细胞中Prkn/parkin的表达。我们发现shPrkn中的 MEC 分化受损细胞，这意味着 MEC 分化需要 PRKN。这些研究表明通过程序化的线粒体自噬对 MEC 分化进行了新的调节，并为未来研究与乳腺线粒体功能相关的发育和疾病奠定了基础。

缩写：AA：抗霉素 A；ATG5：自噬相关5；BAF：巴弗洛霉素 A ₁; BNIP3：BCL2 相互作用蛋白 3；BNIP3L/NIX：BCL2 相互作用蛋白 3 样；COX8A：细胞色素 c 氧化酶亚基 8A；CQ：氯喹；CSN2：酪蛋白β；ECAR：细胞外酸化率；FCCP：三氟甲氧基羰基氰苯腙；FUNDC1：FUN14 域包含 1；HIF1A：缺氧诱导因子1亚基α；L1：泌乳第1天；MAP1LC3B：微管相关蛋白 1 轻链 3 β；MEC：乳腺上皮细胞；mitoQ：米托喹诺；mROS：线粒体活性氧；OCR：耗氧率；P：启动；P16：怀孕第16天；PARP1：聚（ADP-核糖）聚合酶 1；PINK1：PTEN 诱导激酶 1；PPARGC1A：PPARG 共激活剂 1 α；PRKN：parkin RBR E3泛素蛋白连接酶；shNT：短发夹非靶向控制；SQSTM1：螯合体 1；STAT3：信号转导和转录激活因子 3；TEM：透射电子显微镜；TFAM：转录因子 A，线粒体；U：无差别。