Lin Yueh-Chien, Huang Yuan-Li, Wang Ming-Hua, Chen Chih-Yu, Chen Wei-Min, Weng Yi-Cheng, Wu Pei-Yi
Vascular Biology Program, Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Department of Medical Laboratory Science and Biotechnology, Asia University, Taichung 41354, Taiwan.
Biomedicines. 2022 Mar 11;10(3):646. doi: 10.3390/biomedicines10030646.
Prostate cancer (PCa) is the major cause of cancer-related death among aging men worldwide. Recent studies have suggested that calreticulin (CRT), a multifunctional chaperon protein, may play an important role in the regulation of PCa tumorigenesis and progression. However, the underlying mechanisms are still unclear. Integrin is an important regulator of cancer metastasis. Our previous study demonstrated that in J82 bladder cancer cells, CRT affects integrin activity through FUBP-1-FUT-1-dependent fucosylation, rather than directly affecting the expression of β1-integrin itself. However, whether this regulatory mechanism is conserved among different cell types remains to be determined. Herein, we attempted to determine the effects of CRT on β1-integrin in human prostate cancer PC-3 cells. CRT expression was suppressed in PC-3 cells through siRNA treatment, and then the expression levels of FUT-1 and β1-integrin were monitored through RT-PCR. We found that knockdown of CRT expression in PC-3 cells significantly affected the expression of β1-integrin itself. In addition, the lower expression level of β1-integrin was due to affecting the mRNA stability. In contrast, FUT-1 expression level was not affected by knockdown of CRT. These results strongly suggested that CRT regulates cellular behavior differently in different cell types. We further confirmed that CRT directly binds to the 3'UTR of β1-integrin mRNA by EMSA and therefore affects its stability. The suppression of CRT expression also affects PC-3 cell adhesion to type I collagen substrate. In addition, the levels of total and activated β1-integrin expressed on cell surface were both significantly suppressed by CRT knockdown. Furthermore, the intracellular distribution of β1-integrin was also affected by lowering the expression of CRT. This change in distribution is not lysosomal nor proteosomal pathway-dependent. The treatment of fucosydase significantly affected the activation of surface β1-integrin, which is conserved among different cell types. These results suggested that CRT affects the expression of β1-integrin through distinct regulatory mechanisms.
前列腺癌（PCa）是全球老年男性癌症相关死亡的主要原因。最近的研究表明，钙网蛋白（CRT），一种多功能伴侣蛋白，可能在前列腺癌的肿瘤发生和进展调控中发挥重要作用。然而，其潜在机制仍不清楚。整合素是癌症转移的重要调节因子。我们之前的研究表明，在J82膀胱癌细胞中，CRT通过FUBP - 1 - FUT - 1依赖性岩藻糖基化影响整合素活性，而不是直接影响β1整合素本身的表达。然而，这种调节机制在不同细胞类型中是否保守仍有待确定。在此，我们试图确定CRT对人前列腺癌PC - 3细胞中β1整合素的影响。通过siRNA处理抑制PC - 3细胞中的CRT表达，然后通过RT - PCR监测FUT - 1和β1整合素的表达水平。我们发现，PC - 3细胞中CRT表达的敲低显著影响β1整合素本身的表达。此外，β1整合素表达水平较低是由于影响了mRNA稳定性。相反，FUT - 1表达水平不受CRT敲低的影响。这些结果强烈表明，CRT在不同细胞类型中对细胞行为的调节方式不同。我们进一步通过电泳迁移率变动分析（EMSA）证实，CRT直接与β1整合素mRNA的3'非翻译区（3'UTR）结合，因此影响其稳定性。CRT表达的抑制也影响PC - 3细胞对I型胶原底物的粘附。此外，细胞表面表达的总β1整合素和活化β1整合素水平均被CRT敲低显著抑制。此外，β1整合素的细胞内分布也受到CRT表达降低的影响。这种分布变化不依赖于溶酶体或蛋白酶体途径。岩藻糖苷酶处理显著影响表面β1整合素的活化，这在不同细胞类型中是保守的。这些结果表明，CRT通过不同的调节机制影响β1整合素的表达。
