These results were confirmed through a range of experimental models: ES cell lines expressing endogenous levels of -arr1 and MEF cells lacking -arr1 or expressing IGF-1R unable to bind -arr1, all confirming CP-induced ERK activation relies on functional -arr1 interaction with the IGF-1R. paradox in a panel of ES cell lines and found their sensitivity to CP was unaffected by presence of IGF-1, countering a ligand blocking mechanism. CP induced IGF-1R/-arrestin1 association with dual functional outcome: receptor ubiquitination and degradation and decrease in cell viability and -arrestin1Cdependent ERK signaling activation. Controlled -arrestin1 suppression initially enhanced CP resistance. This effect was mitigated on further -arrestin1 decrease, due to loss of CP-induced ERK activation. Confirming this, the ERK1/2 inhibitor U0126 increased sensitivity to CP. Combined, these results reveal the Rabbit polyclonal to MTOR mechanism of CP-induced receptor down-regulation and characteristics that functionally qualify a prototypical antagonist as an IGF-1RCbiased agonist: -arrestin1 recruitment to IGF-1R as the underlying mechanism for ERK signaling activation and receptor down-regulation. We further confirmed the consequences of -arrestin1 regulation on cell sensitivity to CP and demonstrated a therapeutic strategy to enhance response. Defining and suppressing such biased signaling represents a practical therapeutic strategy to enhance response to anti-IGF-1R therapies. 0.05, ** 0.01, ** 0.001. Mechanism of CP-Induced IGF-1R Down-Regulation: -Arrestin1 Recruitment and Receptor Ubiquitination. The next experiments were designed to investigate in detail the CP effects on IGF-1R down-regulation. To avoid the competition between CP and IGF-1 normally present in serum, all experiments were performed in serum-free media (SFM). ES cell lines, serum starved for 12 h, were treated with CP concentrations of 100 ng/mL or 1 g/mL for 24 h, and cell lysates were analyzed for IGF-1R expression using GAPDH as a loading control. As shown in Fig. 2and and and and were treated with 100 ng/mL CP for 48 h. Numbers of viable cells are displayed as percentage of mock transfected, unstimulated control. Data correspond to the mean SEM from three independent experiments. CP-Induced -Arrestin1CMediated IGF-1R ERK Signaling Activation. Previous reports demonstrated -arr1 as a mediator of IGF-1R signaling and cell cycle progression (32); therefore, in the next experiments, we explored the possible agonistic properties of CP, secondary to -arr1 recruitment. The roles of CP on IGF-1R signaling in ES cells were investigated by close monitoring of the dynamics of IGF-1C or CP-mediated activation of the two key downstream IGF-1R signaling pathways, the Ras/Raf/mitogen activated protein kinase kinase (MEK)/ERK pathway and the PI3K/AKT pathway, after short time stimulation. Serum-starved cells were stimulated with IGF-1 or CP (molar concentration of CP ~10-fold less than IGF-1), for up to 60 min before analyzing by WB. On IGF-1 stimulation, the IGF-1R activation loop was phosphorylated within 2 min, demonstrating an increase in its TAK 259 kinase activity. Consequently, both main downstream signaling pathways were activated as demonstrated by ERK and AKT phosphorylation (Fig. TAK 259 5were treated without or with 100 ng/mL CP for 48 h, and the cell viability was assayed by PrestoBlue reagent. The inhibition ratio (quotient between CP-treated and CP-untreated cells) was calculated for each doxycycline dose and displayed as percentage of CP-untreated cells. Data TAK 259 correspond to the mean SEM from three independent experiments. During the experiment, cell lysates were collected at 10 min after CP stimulation and analyzed by WB for P-ERK and total ERK as a loading control, and at 12 h after CP stimulation and analyzed TAK 259 by WB for IGF-1R and GAPDH as a loading control. Signals were quantified by densitometry, normalized to the loading control, and presented as a percentage of the CP-untreated cells. Data correspond to the mean SEM from three independent experiments. ( em C /em ) Indicated cells were pretreated for 60 min without or with the ERK inhibitor U0126, stimulated without or with 100 ng/mL CP. The cell viability assayed 48 h after CP treatment is displayed relative to untreated control. Data correspond to the mean SEM from three independent experiments. The percentage inhibition of CP-treated cells is displayed relative to CP-untreated cells. The proportion of cells inhibited by CP treatment at T48h, described by the CP ratio (CP treated versus untreated) confirmed our hypothesis, showing that down to a certain level of -arr1, the cells are more resistant.
