Functional Specificity of the Members of the Sos Family of Ras-GEF Activators: Novel Role of Sos2 in Control of Epidermal Stem Cell Homeostasis.

Fernando C Baltanás,Rocío Fuentes-Mateos,Rósula García-Navas,Natalia Fernández-Parejo,Cynthia Mucientes-Valdivieso,L Francisco Lorenzo-Martín,Eugenio Santos,Nuria Calzada,Carmen Segrelles,Jesús M Paramio,Xosé R Bustelo

doi:10.3390/cancers13092152

Abstract

Simple SummaryThe Sos Ras-GEFs are known to participate in a wide range of skin-related diseases including cutaneous cancers, cardio-facio-cutaneous syndromes, or hirsutism. However, the specific functional roles played by the Sos1 and/or Sos2 family members in specific skin compartments remain largely unknown. This report aimed at precisely characterizing the specific functions played by Sos1 and/or Sos2 in keratinocytes, an essential cellular component of the skin. Our data show that Sos1 and Sos2 make overlapping contributions to both keratinocyte proliferation and survival. However, Sos1 seems to have a preferential involvement in regulating the ERK axis, whereas Sos2 seems to control the signaling output from the PI3K axis. We also uncovered an essential role of Sos2 in the control of the population of epidermal stem cells.Prior reports showed the critical requirement of Sos1 for epithelial carcinogenesis, but the specific functionalities of the homologous Sos1 and Sos2 GEFs in skin homeostasis and tumorigenesis remain unclear. Here, we characterize specific mechanistic roles played by Sos1 or Sos2 in primary mouse keratinocytes (a prevalent skin cell lineage) under different experimental conditions. Functional analyses of actively growing primary keratinocytes of relevant genotypes—WT, Sos1-KO, Sos2-KO, and Sos1/2-DKO—revealed a prevalent role of Sos1 regarding transcriptional regulation and control of RAS activation and mechanistic overlapping of Sos1 and Sos2 regarding cell proliferation and survival, with dominant contribution of Sos1 to the RAS-ERK axis and Sos2 to the RAS-PI3K/AKT axis. Sos1/2-DKO keratinocytes could not grow under 3D culture conditions, but single Sos1-KO and Sos2-KO keratinocytes were able to form pseudoepidermis structures that showed disorganized layer structure, reduced proliferation, and increased apoptosis in comparison with WT 3D cultures. Remarkably, analysis of the skin of both newborn and adult Sos2-KO mice uncovered a significant reduction of the population of stem cells located in hair follicles. These data confirm that Sos1 and Sos2 play specific, cell-autonomous functions in primary keratinocytes and reveal a novel, essential role of Sos2 in control of epidermal stem cell homeostasis.

Highlights

RAS proteins are critical signal transduction regulators that regulate cell proliferation, differentiation, migration, and survival in different cell types [1,2,3]
Since tamoxifen appears to affect the viability of keratinocytes in culture even at low concentrations, we decided to test the efficiency of GFP-Adeno-Cre infection as an alternative method to accomplish Sos1 depletion in freshly isolated Sos1 KO and Sos1/2 KO primary keratinocytes
Under the conditions specified in Materials and Methods, we observed that, after 72 h of adenoviral infection, the presence of the GFP-labeled adenovirus was detected in almost all primary keratinocytes in the culture (93%), and Western blot analysis of the GFP-Adeno-Cre-infected keratinocytes (Sos1 KO and Sos1/2 DKO) showed practically complete disappearance of Sos1 protein expression (Figure S1A–C)

Summary

Introduction

RAS proteins are critical signal transduction regulators that regulate cell proliferation, differentiation, migration, and survival in different cell types [1,2,3]. Sos and Sos form the most widely expressed and functionally relevant family of GEFs involved in activation of mammalian RAS proteins by upstream cellular signals [4,5,6,7]. Despite their similar protein structures and cellular expression patterns, a variety of specific functionalities have been reported for Sos and Sos in different biological contexts [7]. The development of a conditional tamoxifen-inducible Sos1-null mouse model allowed bypassing the embryonal lethality of Sos1-null mutants and testing of the functional specificity/redundancy of Sos and Sos in adult animals and tissues [11]. It is apparent that additional comprehensive functional studies of specific tissue/cell lineages derived from KO mouse models are needed to fully understand the specific functional roles of Sos and Sos in different physiological or pathological contexts

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