349 Regulation of epithelial stem cell proliferation and differentiation by the Gαi heterotrimeric G protein

Abstract

Regulation of cAMP signaling in the skin and other organs plays a central role in the regulation of cell differentiation and tumor formation. Decrease in cAMP signaling as a result of Gαs or protein kinase A (PKA) inactivation causes an expansion of basal progenitor keratinocytes in the mouse skin, resulting in basal cell carcinoma formation (Iglesias-Bartolome et al, Nature Cell Biology, 17:p793, 2015). These observations suggest that the regulation of cAMP levels is potentially involved in epithelial stem cell fate decisions. We hypothesized that activation of Gαi subunit, which opposes Gαs signaling by reducing cAMP production, might participate in this regulatory network. To study Gαi signaling we took advantage of a synthetic biology approach by using a modified human muscarinic receptor coupled to Gαi (hM4Di) which is exclusively activated by the synthetic ligand clozapine N-oxide (CNO). By expressing and activating hM4Di in primary keratinocytes we demonstrated that the activation of Gαi induces cell proliferation and decreases differentiation. Moreover, treatment with CNO in organotypic cultures led to an increase in skin thickness and cell proliferation. This effect was rescued when the cells were treated with the Gαi inhibitor pertussis toxin (PTX). We next validated these results using a tetracycline inducible mouse model. We targeted the expression of hM4Di to the basal/stem cell compartment of the skin and induced its activation by treating with CNO. Analysis of skin histology reveals that Gαi activation results in skin hyperplasia, with expansion of the K5+ basal compartment and increase of the proliferation marker PCNA. Mechanistically, activation of Gαi in keratinocytes leads to increase in ERK and AKT phosphorylation. Our findings indicate that signaling through Gαi and its yet to be identified coupled GPCRs could play a central role in the regulation of keratinocytes differentiation and proliferation.