Molecular mechanisms of PI3K isoform dependence in embryonic growth

Abstract

Objective: The phosphoinositide 3-kinase (PI3K) pathway is an important signaling mechanism for cell proliferation and metabolism. Mutations that activate PIK3CA may make cells p110α dependent, but when phosphatase tensin homolog (PTEN) is lost, the p110β isoform of PI3Ks becomes more important. However, the exact mechanism underlying the prevalence of p110s remains unclear. In this study, our aim was to elucidate the processes behind PI3K isoform dependency in a cellular model of embryonic development.

Material and methods: In order to understand PI3K isoform prevalence, mouse embryonic fibroblasts (MEFs) were used and p110β, PTEN and Rac1 activity was modulated using retroviral plasmids. Expression levels and cellular growth were assessed by performing immunoblots and crystal violet assays.

Results: The levels of PTEN had only a partial effect on the prevalence of PI3K isoforms in MEFs. The dependency on p110α diminished when PTEN was depleted. Of note, when PTEN expression was repressed, there was no full transition in dependency from one PI3K isoform to the other. Interestingly, the viability of PTEN-depleted MEFs became less dependent on p110α and more dependent on p110β when p110β was overexpressed. Nevertheless, the overexpression of p110β in conjunction with PTEN knock-downs did not result in a complete shift of isoforms in PI3Ks. Finally, we investigated Rac1 activation with a mutant allele and determined a more potent increase in p110β prominence in MEFs.

Conclusion: These findings suggest that multiple cellular parameters, including PTEN status, PI3K isoform levels, and Rac1 activity, combine to influence PI3K isoform prevalence, rather than a single determinant.

Keywords: PI3K isoform prominence; PTEN; Rac1; mouse embryonic fibroblasts.

Figure 1

Molecular characterization of PI3K isoform prevalence in MEFs. (A) MEFs were treated with varying doses of p110α and p110β specific inhibitors. Crystal violet assays were performed to determine the cellular proliferative capacity. (B) PTEN was knocked-down in MEFs using shRNA constructs. Knock-down efficiency was determined in an immunoblot using anti-PTEN antibodies. pAkt and pS6 antibodies were used to assess the level of PI3K activation whereas anti-actin were used as loading control. (C) PTEN knocked-down MEFs were treated with varying doses of p110α and p110β specific inhibitors. Crystal violet assays were performed to determine the cellular proliferative capacity. (D) Control or PTEN knocked down MEFs were treated either with DMSO or isoform specific PI3K inhibitors, BYL-719 (BYL) and KIN-193 (KIN), specific for p110α and p110β or GDC, a pan-PI3K inhibitor respectively. The efficacy of PI3K signaling in these cells was assessed using immunoblots for p-Akt (T308 and S473) and p-S6 (S235/236). Actin served as the control for loading. *p<0.05, ***p<0.005, (the error bars in the graphs represent the standard deviation, and the experiments were conducted independently three times) MEFs: Mouse embryonic fibroblasts, PTEN: Phosphatase tensin homolog

Figure 2

PTEN loss in combination with an increase in PIK3CB expression promote an elevated dependence on p110β. (A) Overexpression of p110β is achieved through retroviral expression of PIK3CB in MEFs. (B) Isoform specific PI3K inhbitors were applied to wt or p110β overexpressing MEFs, crystal violet assays were performed. (C) An increased amount of isoform specific PI3K inhbitors as well as pan-PI3K (GDC) and pan-AKT (MK2206) inhibitors were applied to wt or p110β overexpressing MEFs, crystal violet assays were performed. (D) The shPTEN1 and 2 MEFs were subjected to two rounds of AdCre treatments, and their proliferation rates were assessed using the crystal violet test. As a negative control for PI3K knockdown, LacZ expressing adenoviruses were used. The spectrometric quantification of crystal violet staining data was performed for MEFs treated with adenovirus. (E) The cellular viability of p110β overexpressing shPTEN-MEFs treated with BYL719 and KIN193 was measured. (F) Crystal violet assays for shPTEN MEFs treated with higher concentrations of BYL719 and KIN193 were quantified. **p<0.01, ***p<0.005, ****p<0.0001, ns: Not significant, p>0.05) (the error bars in the graphs represent the standard deviation, and the experiments were conducted independently three times) MEFs: Mouse embryonic fibroblasts, PTEN: Phosphatase tensin homolog

Figure 3

Figure 3. Activated Rac1 expression tilts the balance towards increased p110β prominence. (A) MEFs expressing mutant Rac1 versions were analyzed for expression of the depicted proteins and phosphor-proteins in immunoblots. Actin served as the control for loading. (B) Either empty vector control or mutant Rac1 expressing MEFs were treated with moderate doses of p110α and p110β specific inhibitors (BYL, KIN). Crystal violet assays were performed to determine the cellular proliferative capacity. **p<0.01, (the error bars in the graphs represent the standard deviation, and the experiments were conducted independently three times) MEFs: Mouse embryonic fibroblasts