PI3K/Akt responses to oxytocin stimulation in Caco2BB gut cells

Abstract

Recently, we discovered oxytocin receptor (OTR) expression in the developing gut villus epithelium that emerges in villus-crypt junctions after weaning. Oxytocin (OT) and OTR regulate many physiological functions in various tissues; however, their function in gut epithelium is unknown. We explored responses of PI3K and Akt phosphoisoforms to OT stimuli in the Caco2BB human gut cell line. In Caco2BB cells, PI3K and pAkt levels peaked at 62.5 nM OT. At higher concentrations, PI3K decreased more gradually than pAkt(S473) suggesting that the pAkt(S473) response is separate from PI3K. At ≤7.8 nM OT, pAkt(T308) increased while pAkt(S473) decreased. Using a specific OTR antagonist, we demonstrated that responses of pAkt(T308) to OT depend on OTR in contrast to the partial OTR-dependence of the pAkt(S473) response. Differential pAkt phosphoisoform responses included pAkt phosphoserine 473 persistently free of phosphothreonine 308. The reduction in PI3K after 62.5 nM OT for 30 min coincided with OTR internalization. The PI3K/Akt activation profile was somewhat different in other cell lines (MCF-7 breast cancer cells, HT29 gut cells), which have PI3K activating mutations, that were examined to establish experimental parameters. In Caco2BB cells, the divergent effects of OT upon pAkt phosphoisoforms suggests separate sub-pathways; pAkt (T308) activation depends on OTR via the PI3K pathway and pAkt(S473) presumably results from its specific kinase mTORC2 (mammalian target of rapamycin complex 2). Thus, OT may modulate gut cell functions downstream of mTOR complexes (e.g., translation control as suggested by others in uterine cells). We will next explore OT-stimulated kinase activities downstream of mTOR related to pAkt phosphoisoforms.

Calming Cycle Theory and the Co-Regulation of Oxytocin

“…At the cellular level, in vitro experiments using a gut cell line have demonstrated that OT modulates important molecules in the stress signalling pathway (Klein, Tamir, Hirschberg, Glickstein, & Welch, 2013;Klein, Tamir, & Welch, 2011) and plays a regulatory role in translation of proteins (Klein et al, 2014). These studies suggest that biochemical factors in milk, including exogenous OT from the mother, play an important role in the unfolded protein response to reduce cellular stress.…”

Oxytocin modulates markers of the unfolded protein response in Caco2BB gut cells

“…Subsequently, we found that OT attenuated the stimulation of the PI3K/Akt pathway induced by fresh growth medium (FGM) (Klein et al 2013). OT also modulated the phosphatase and tensin homolog (PTEN) (Klein et al 2011) and the mammalian target of rapamycin (mTOR) pathway (Klein et al 2013). Several of these OTR signal transducers are associated with both ASD and protein translation; mutations in PI3K, PTEN, and tuberosclerosis protein (TSC2), members of the Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway that control ribosomal protein and translation factor activity have been linked with ASD (Kelleher and Bear 2008).…”

“…We analyzed the response of the mTORC1 substrate 4E-BP1, a negative regulator of eIF4E, to test whether OT influences translation factors downstream of mTORC1. For all experiments, we have defined high (62.5 nM) and low (7.8 nM) concentrations of OT based upon convention from our prior work (Klein et al 2011) where high 62.5 nM OT maximally stimulated the Akt/PI3K pathways. Protein extracts from Caco2BB cells treated with high or low concentrations of OT for 30 min were fractionated by capillary electrophoresis and analyzed for phosphorylation.…”

“…To gain insight into the signaling pathway(s) modulated by OT stimulation of the OTR in gut epithelium, we previously explored the response of the phosphoinositol-3 kinase (PI3K)/ Akt pathway to OT in human gut epithelial cell lines (Klein et al 2011). We found that OT modulated the abundance of pAkt T308 via PI3K in a dose-dependent manner; notably, 62.5 nM OT maximally stimulated the Akt/PI3K pathways in comparison with higher (up to 1,000 nM OT) or lower OT concentrations.…”

Intracellular Pathways Associated with the Etiology of Autism

“…Recent studies suggest that, in addition to its role in the brain, OXT may also have an important neuromodulatory role in the gut by activating the PI3K/Akt pathway in a dose-and time-dependent manner [164]. Our lab has found decreased plasma oxytocin in individuals with autism, and these oxytocin levels correlated with high mTOR, suggesting a possible relationship between oxytocin and aberrant Akt pathway signaling [128].…”