The Study Of The Effect Of Interaction Between Hormones/growth Factors On PI3K/AKT Signaling Pathway And Its Mechanism

Background and aims:The phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)signaling pathway is a central controller of cell growth,proliferation,metabolism,survival,and angiogenesis and it is a key driver for cancer development[1-3].The AKT is the key molecule in this pathway.Activated AKT can activated hunsdreds of substrates,regulating a variety of cellular functions including growth,proliferation,metabolism,apoptosis,and angiogenesis[4-7].Aberrant up-regulation of PI3K-AKT pathway leads to cancers,while inhibition of PI3K/AKT signaling is associated with insulin resistance diseases.There are many hormones and growth factors in our body,and many of them including insulin,growth hormone,epidermal growth factor,hepatocyte growth factor,can activate PI3K-AKT signaling.However,our body keeps health.The mechanism underling the signaling homeostasis is largely unknown.Therefore it is important to clarify the mechanism for the prevention and treatment of cancer and diabetes.Our group previously reported that the association between PTEN and PI3K p85 subunit plays an important role in keeping the PI3K/AKT signaling homeostasis under the condition of GH-mediated insulin resistance and the acute alcohol exposure.We now aim to study the effect of interaction between hormones/growth factors on PI3K/AKT signaling and its mechanism,clarifying the signaling homeostasis mechanism.Methods1.The effect of interaction between hormones/growth factors on the PI3K/AKT signaling pathway in vivo.To study the effect of interaction between HGF-INS,EGF-INS,GH-EGF,healty male balb/c mice were divided into 2 groups.Mice were injected intraperitoneally with the first hormone/growth factor(HGF,EGF,GH)3 hour or 30 minute before sacrifice,and injected with the second hormone/growth factor(insulin,insulin,EGF,respectively).Western blot and/or Immunofluorescence were performed to test the p-AKT level in the liver.2.The effect of interaction between hormones/growth factors on the PI3K/AKT signaling pathway in vitro.HepG2 cells and HEK 293T cells were treated with the first hormone/growth factor(HGF,EGF,GH)or vehicle(control group)30 minutes or 4 hours before lysis and the second hormone/growth factor(insulin,insulin,EGF,respectively)10 minutes before lysis.Western blot and/or Immunofluorescence were performed to test the p-AKT level in the cells3.Molecular mechanism of the effect of interaction between hormones/growth factors on the PI3K/AKT signaling pathway.After transfected with the PTEN mutant C124S or G129E plasmid or p50 plasmid(N terminal deletion of PI3K p85 subunit),HepG2 cells and HEK 293T cells were treated with the first hormone/growth factor(HGF,EGF,GH)under different duration of time(30 minutes or 4 hours)and with the second hormone/growth factor(insulin,insulin,EGF,respectively).Western blot and/or immunofluorescence were performed to test the p-AKT level in the cells.Results1.Prolonged pre-stimulation of Hormones/Growth factors Inhibits the Second Hormones/Growth factors-induced AKT activity in vivo.Western blot and/or immunofluorescence results showed that after 3 hour pre-stimulation of the first hormone/growth factor,the expression of p-AKT in liver induced by the second hormones/growth factors was significantly decreased in vivo;2.Prolonged pre-stimulation of Hormones/Growth factors Inhibits the Second Hormones/Growth factors-induced AKT activity in vitro.Western blot and/or immunofluorescence results showed that after 4 hours pre-stimulation of the first hormone/growth factor,the expression of p-AKT induced by the second hormones/growth factors was significantly decreased in HepG2 cells and 293T cells;While no significant differences were observed in the expression of p-AKT induced by the second hormones/growth factors after 30 minutes pre-stimulation of the first hormone/growth factor.3.Mutant PTEN impairs the inhibition in AKT activation between hormones or growth factors in vitro.Western blot and/or immunofluorescence results showed that no significant differences were observed in the expression of p-AKT between control group,30min group and 4 hour group after transfected with C124S or G129E plasmid.4.PI3K p85 subunit is associated with the hormone-hormone inhibition in vitro.Western blot results showed that no significant differences were observed in the expression of p-AKT between control group,30min group and 4 hour group after transfected with p50 plasmid.ConclusionsThe effect of interaction between hormones/growth factors of 3 different combinations involving 4 hormones or growth factors on the PI3K/AKT signaling pathway in different cell lines and in animals was investigated in our study.We found that prolonged pre-stimulation of a first hormone/growth factor inhibited the second hormone/growth factor-mediated AKT activation,while short time pre-stimulation of a first hormone/growth factor had no effect on the second hormone/growth factor-mediated AKT activation,which may be the reason why the PI3K/AKT signaling pathway kept balanced under the stimulation of a viarety of hormones and growth factors.Our study also found that PTEN and PI3K p85 subunit were key negative regulators in the inhibition effect between hormones/growth factors,and inhibition effect might be closely associated with the interaction between PTEN and p85.