Detection of IGF-1R expression
The expression pattern of IGF-1R was evaluated by CLSM (Confocal Laser scanning microscope). As we can see in the Fig. 1A, CLSM analysis showed IR was mainly in the cell membrane, and a small number of insulin receptors were expressed in the cytoplasm (Fig. 1B). Furthermore, Flow cytometry also showed that A549 cell expressed high level of IGF-1R.
In addition, we knocked down IGF-1R through the SiRNA method, and the results showed that the expression level of IGF-1R was significantly reduced (knocking down IGF-1R did not cause significant cell apoptosis) (Fig. 1C).
Figure 1A. Detection of IR expression by CLSM (A) and flow cytometry (B). C. IGF-1R was knock-down through the use of siRNA technology. Data are expressed as the mean ± standard deviation. P < 0.05 was considered to indicate a statistically significant difference.
Internalization of IGF-1 in A549 cell
The internalization of insulin/IGF-1 on A549 cell was checked. The cells were challenged with IGF-1 for different time points. The cell samples were then analyzed by Confocal Laser Scanning Microscope (CLSM). The results showed that fluorescently labeled-insulin was internalized into A549 cell in a time-dependent manner (Figure.2A). Additionally, IGF-1’s internalization was also evaluated CLSM (Figure. 2B).
Figure 2A. The internalization dynamics of insulin on A549 cells. B. The internalization dynamics of IGF-1 on A549 cells. Data are expressed as the mean ± standard deviation. P < 0.05 was considered to indicate a statistically significant difference.
Intracellular trafficking of IR under insulin but not IGF-1 treatment
Here, we studied the intracellular trafficking of IR under insulin stimulation, and the results indicated that IR could internalize in to cell cytoplasm in a time-dependent manner under insulin treatment. In addition, IR also transported into cell nuclei (Fig. 3A). In addition, we also explored the trafficking of IR under IGF-1 treatment, and results showed that IR could internalize into cell cytoplasm, but IR could not transport into cell nuclei (Fig. 3B). Western-Blot also confirmed this result. These results suggested that the nuclear localization of IR is specifically induced by insulin but not IGF-1. These results suggested that IR’s nuclear localization may exhibit important biological activities in A549 cell nuclei.
Figure 3A. The internalization dynamics of IR under insulin treatment. B. The internalization dynamics of under IGF-1 treatment. Data are expressed as the mean ± standard deviation. Data are expressed as the mean ± standard deviation. P < 0.05 was considered to indicate a statistically significant difference.
Clathrin and caveolin are involved in IR’s Endocytosis
The above study has showed that IGF-1/IGF-1R could internalize into cell cytoplasm. Here, the endocytic mechanism of IR was explored. Study has shown that the endocytic pathway of the same cytokine/receptor is different in different types of cells. In lung cancer cell, the endocytic pathway of the insulin/IR remains unclear. Clathrin-dependent endocytosis or caveolin-mediated endocytosis is involved in the endocytosis of cytokine/growth factor. Additionally, the non-clathrin- and caveolin-dependent pathway was also existed. For this, co-localization analysis was conducted to study the IR’s endocytic pathway. The co-localization signals between caveolin/IR and clathrin/IR were detected under insulin treatment, which suggested that both clathrin and caveolin were involved in the IR’s endocytosis (Fig. 4A). In addition to insulin, we also analyzed the endocytic mechanism of IR under the stimulation of IGF-1 (Fig. 4B).
Figure 4. A. Both clathrin and caveolin were involved in the IR’s endocytosis under insulin treatment. B. Both clathrin and caveolin were involved in the IR’s endocytosis under IGF-1 treatment. P < 0.05 was considered to indicate a statistically significant difference.
The internalized IR localized in different types of endosomes
We further analyzed which types of endosomes IR enter into by CLSM. The colocalization signal of IR and EEA1 (early endosome marker) could be detected, which indicated that IR enters into the early endosome (Fig. 5). It is well-known that the recycling endosome is rich in Rab4 and Rab11, whereas Rab7 and Rab9-positive endosome is rich in the late endosomes. Colocalization analyses showed that IR was localized in Rab5/7/11-positive endosome, which provides an explanation for different cytoplasmic localization of IR (Different types of endosomes could transport IR to different destinations).
Figure 5. A. The IR transported into different types of endosomes under IGF-1 treatment. P < 0.05 was considered to indicate a statistically significant difference.
Tyrosine phosphorylation of IR is required for nuclear localization of IR
We test whether the IR’s nuclear localization is associated with IR’s tyrosine phosphorylation. We used HNMPA-(AM)3 (Insulin receptor tyrosine kinase inhibitor) to treat the cells, and the results showed that the nuclear localization of IR was inhibited, but its internalization has not been affected (Fig. 6). This finding suggests that IR phosphorylation is required for IR’s nuclear localization.
Figure 6. Tyrosine phosphorylation of IR is required for IR’s nuclear localization. The experimental process has been described in detail in the materials and methods section. P < 0.05 was considered to indicate a statistically significant difference.
Nup358 is involved in IR’s nuclear localization
Crossing the nuclear membrane is the most important step in the process of IR’s nuclear translocation. Previous studies have shown that NUP358 plays an important role in the nuclear transport process of IGF-1R 9. IGF-1R has a similar structure to IR, and both IGF-1 and insulin can bind IGF-1R. Similarly, IGF-1 and insulin can also bind to IR. Therefore, we analyzed whether NUP358 is involved in IR’s nuclear translocation. As shown in Fig. 7A, the colocalization analyses indicated that IR can interact with Nup358, and the results form IP-WB also indicated that NUP358 interacted with IR.
In order to further determine the role of NUP358 in IR’s nuclear transport, NUP358 was knocked down using the SiRNA method (Fig. 7B). The results showed that the nuclear localization of IR was significantly reduced. This further confirmed that NUP358 was involved in the nuclear localization of IR (Fig. 7C).
Figure 7. A. Detection of colocalization between IR and UNP358. B. Analysis of interactions between IR and UNP358 by IP-WB.C. NUP358 Knocking down inhibited IR’s nuclear localization. The experimental process has been described in detail in the materials and methods section. P < 0.05 was considered to indicate a statistically significant difference.
IR nuclear localization is associated with cell proliferation of A549
To explore the function or role of nuclear-localized IGF-1R, a model of the non-nuclear-localized IR was established by knocking down Nup358 (NUP 358-knock-down does not affect the proliferation of lung cancer cells, and did not lead to the apoptosis of lung cancer cells (Data not shown)), and the results indicated that IR’s nuclear localization was significantly reduced compared to control (Figure.7C), but IR’s internalization was not affected. Then MTT experiments were used to evaluate the role of nuclear-localized IR. As shown in Fig. 8A, the 549-cell proliferation ability was reduced compared to the control. In order to further evaluate the role of nuclear-localized IR, we used a nuclear export inhibitor (Leptomyocin B, which can increase the residence time of IR in cell nuclei), and the results showed that the cell's proliferation ability was also increased (Fig. 8B). To further analyze the effect of nuclear-localized IR on A549 cell proliferation, cell cycle was determined by Flow cytometry, and the results showed that the proportion of cells in S phase was significantly increased compared to the control (Fig. 8C). In addition, Ki67 expression was also enhanced compared to control (Fig. 8D).
Figure 8. A. Nuclear-localized IR is closely related to the proliferation of A549 cells. B. Cell's proliferation ability was also enhanced by increasing the IR’s residence time in the cell nuclei. C. IR’s nuclear localization was associated with cell cycle. D. Ki67 expression was enhanced. Data are expressed as the mean ± standard deviation. P < 0.05 was considered to indicate a statistically significant difference.
Nuclear-localized IGF-1R increased the nuclear retention of signaling molecule
We further explored the potential mechanism by which the nuclear-localized IR promote cell proliferation. Since the biological activity of insulin/IR is achieved by IR-mediated signal transduction, we explored the mechanism of the action of nuclear-localized insulin/IR from the perspective of IR-mediated signaling. As indicated in Fig. 9A, the results showed that the activation of p-ERK1/2 was significantly prolonged and increased compared to the non-nuclear-localized IR group, which suggested that nuclear-localized IR still has the ability to trigger downstream signals. But, insulin-induced other signaling pathways was not affected (Fig. 9B). This may be one of the potential effects of nuclear-localized IR.
Figure 9. A. The nuclear localization of IR is closely related to the activation of ERK1/2. The experiment process has been described in detail in the materials and methods section. Data are expressed as the mean ± standard deviation. P < 0.05 was considered to indicate a statistically significant difference.