Department of Physiology and Biophysics, Weill Cornell Medicine in Qatar, Doha, Qatar.
Department of Biochemistry, Weill Cornell Medicine in Qatar, Doha, Qatar.
Biochim Biophys Acta Mol Cell Res. 2019 Apr;1866(4):727-736. doi: 10.1016/j.bbamcr.2018.12.003. Epub 2018 Dec 5.
Calreticulin an endoplasmic reticulum (ER) chaperone that is involved in the quality control process and plays an important role as a regulator of intracellular calcium homeostasis. Previously, we illustrated that loss of calreticulin (crt-/-) results in the activation of ubiquitin-proteasome pathway facilitating the increased resistance to apoptosis. Our preliminary data illustrated a significant increase in the endocytosis in the calreticulin knockout mouse embryonic fibroblast cells (crt-/-). Therefore, we hypothesized that the mechanism for this increased endocytosis in the crt-/- cells is due to onset of ER stress. To test this hypothesis, we measured endocytosis in the wild type (wt) and crt-/- cells using uptake of fluorescent dextran and showed a significant increase in the rate of its uptake in crt-/- cells as compared to wt cells. To determine the endocytic pathway involved we examined both clathrin and caveolin-1 dependent endocytosis. Our results illustrated no change in the expression of clathrin heavy chain while there was a significant increase in the expression of caveolin-1 in the crt-/- cells as compared to the wt cells. Furthermore, using shRNA we illustrated that knockdown of clathrin heavy chain had no effect on endocytosis in the crt-/- cells. While knock-down of caveolin-1 significantly reduced endocytosis in the crt-/- cells. Finally, we illustrated that a chemical chaperone, 4‑phenylbutyrate significantly reduced both the endoplasmic reticulum stress and endocytosis in the crt-/- cells. Our data shows for the first time, that ER stress led to enhanced caveolin-1 mediated endocytosis and reversal of ER stress reduces endocytosis.
钙网织蛋白是内质网（ER）伴侣，参与质量控制过程，作为细胞内钙稳态的调节剂发挥重要作用。先前，我们表明钙网织蛋白（crt-/-）的缺失会激活泛素蛋白酶体途径，从而增加细胞对凋亡的抵抗力。我们的初步数据表明，钙网织蛋白敲除的小鼠胚胎成纤维细胞（crt-/-）中的内吞作用显著增加。因此，我们假设 crt-/-细胞中这种内吞作用增加的机制是由于内质网应激的发生。为了验证这一假设，我们使用荧光葡聚糖摄取来测量野生型（wt）和 crt-/-细胞中的内吞作用，并显示 crt-/-细胞中其摄取率显著高于 wt 细胞。为了确定涉及的内吞途径，我们检查了网格蛋白和 caveolin-1 依赖性内吞作用。我们的结果表明，网格蛋白重链的表达没有变化，而 crt-/-细胞中 caveolin-1 的表达显著增加。此外，通过 shRNA 实验，我们表明，敲低网格蛋白重链对内质网应激诱导的内吞作用没有影响。而 caveolin-1 的敲低则显著降低了 crt-/-细胞中的内吞作用。最后，我们表明，化学伴侣 4-苯丁酸可显著降低 crt-/-细胞中的内质网应激和内吞作用。我们的数据首次表明，内质网应激导致增强的 caveolin-1 介导的内吞作用，而内质网应激的逆转可降低内吞作用。
