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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: J Invest Dermatol. 2019 Nov 7;140(6):1195–1203.e3. doi: 10.1016/j.jid.2019.10.012

Activation of GPCR-Gαi signaling increases keratinocyte proliferation and reduces differentiation, leading to epidermal hyperplasia

M Pilar Pedro 1, Natalia Salinas Parra 1, J Silvio Gutkind 2, Ramiro Iglesias-Bartolome 1
PMCID: PMC7202966  NIHMSID: NIHMS1542207  PMID: 31707029

Abstract

G-protein-coupled receptors (GPCRs) and their associated heterotrimeric G proteins impinge on pathways that control epithelial cell self-renewal and differentiation. While it is known that Gαs signaling regulates skin homeostasis in vivo, the role of GPCR-coupled Gαi proteins in the skin is unclear. Here, by using a chemogenetic approach, we demonstrate that GPCR-Gαi activation can regulate keratinocyte proliferation and differentiation and that overactivation of Gαi-signaling in the basal compartment of the mouse skin can lead to epidermal hyperplasia. Our results expand our understanding of the role of GPCR-cAMP signaling in skin homeostasis and reveal overlapping and divergent roles of the cAMP-regulating heterotrimeric Gαs and Gαi proteins in keratinocytes.

INTRODUCTION

Skin development and homeostasis depends on the activity of specific basal progenitor and stem cell populations (Belokhvostova et al., 2018, Iglesias-Bartolome and Gutkind, 2010). Dysregulation of the balance between self-renewal and differentiation in epithelial cells can lead to the development of numerous pathologies, including aging-related disorders, decreased tissue regeneration and cancer. Studies investigating the molecular factors that govern epithelial cell fate show that G-protein-coupled receptors (GPCRs) and their associated effector cascades impinge on pathways that regulate self-renewal and differentiation (Callihan et al., 2011, Iglesias-Bartolome et al., 2015, Kobayashi et al., 2010, Mo et al., 2014). GPCRs are the largest family of cell-surface molecules responsible for signal transduction, relaying their signaling by coupling to heterotrimeric Gα, β and γ subunits (Pierce et al., 2002). Since GPCRs are the direct or indirect target of more than one third of therapeutic drugs on the market (Santos et al., 2017), they represent a unique potential target for the pharmacological intervention of stem cell activity.

Gα proteins dictate a great degree of the signaling specificity by linking GPCRs to a diverse set of downstream effectors. Both heterotrimeric Gαs and Gαi subfamily members signal by regulating the levels of the intracellular second-messenger cyclic AMP (cAMP), either stimulating (Gαs) or inhibiting (Gαi) the production of cAMP by adenylate cyclases. While it is known that Gαs signaling regulates skin homeostasis in vivo (Iglesias-Bartolome et al., 2015) the role of GPCR-coupled Gαi proteins in the skin is unclear. Gαi proteins in keratinocytes have been implicated primarily in the control of oriented cell division in the epidermis (Williams et al., 2011, Williams et al., 2014). Gαi3 is necessary for the formation of the apical polarity complex, non-planar cell division and differentiation in the developing epidermis in mice (Williams et al., 2014). These functions are dependent on the scaffolding role of GDP-bound Gαi and are not related to GPCR signaling or the regulation of adenylyl cyclases by activated GTP-bound Gαi (Williams et al., 2011, Williams et al., 2014). In contrast, several ligands that activate Gαi-coupled GPCRs have been shown to influence epidermal homeostasis and wound healing, including lysophosphatidic acid (LPA) (Balazs et al., 2001, Piazza et al., 1995), sphingosine-1-phosphate (S1P) (Allende et al., 2013) and leukotriene B4 (LTB4) (Oyoshi et al., 2012). In addition, Gαi can control pathways involved in keratinocyte proliferation and stem cell maintenance, such as hedgehog GLI (Kong et al., 2019, Ogden et al., 2008, Pusapati et al., 2018, Villanueva et al., 2015) and hippo YAP1 (Yu et al., 2012). Therefore, regulation of cAMP levels by GTP-bound Gαi can have functional consequences in the skin, beyond the scaffolding role of GDP-bound Gαi.

Here, by utilizing a chemogenetic approach to modulate Gαi in human keratinocytes and mouse skin, we find that GPCR-Gαi activation can increase keratinocyte proliferation and decrease differentiation, leading to epidermal hyperplasia. These results emphasize the role of cAMP signaling in skin homeostasis and point towards overlapping and divergent roles of the cAMP-regulating heterotrimeric Gαs and Gαi proteins in the modulation of epithelial cell fate.

RESULTS

GPCR-induced Gαi activation in human keratinocytes triggers proliferation and decreases differentiation

To characterize the specific role of Gαi signaling by GPCRs in keratinocytes and to avoid variability of GPCR expression levels and potential autocrine loops, we took advantage of the human muscarinic designer receptor (DREADD) coupled to Gαi (hM4Di) (Urban and Roth, 2015). This receptor is exclusively activated by the synthetic inert ligand clozapine-N-oxide (CNO), allowing to activate specific G-protein signaling without interference from endogenous ligands (Figure 1a). hM4Di activation by CNO leads to reduced levels of intracellular cAMP (Figure S1A). DREADDs have been used extensively in cellular and animal models (Urban and Roth, 2015, Vaque et al., 2013) and are a unique resource to dissect specific heterotrimeric G protein biological functions.

Figure 1: Gαi-activation induces proliferation and reduces differentiation in human keratinocytes.

Figure 1:

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(a) Schematic representation of hM4Di-Gαi activation by CNO but not the endogenous ligand acetylcholine (Ach), and IF showing the N/TERT2G stable cell line expressing HA-tagged hM4Di. (b) Representative picture of EdU-proliferation assay in hM4Di N/TERT2G cells treated with 1 μM CNO (+CNO) or vehicle (DMSO, −CNO). (c) Representative quantification of three independent experiments as detailed in (b) (*p<0.05; t test). (d) Representative qRT-PCR analysis in triplicate showing expression of keratin 5 (KRT5), keratin 1 (KRT1) and transglutaminase 3 (TGM3) in differentiated hM4Di positive N/TERT2G cells, treated (+CNO) or not (-CNO) with CNO. Values are indicated as fold over non-differentiated cells, shown as a red dotted line (****p< 0.0001; t test). (e) Western blot analysis of expression of basal (p63) and differentiation (K10) markers in differentiated hM4Di positive cells treated (+CNO) or not (-CNO) with CNO. (f) IF showing the expression of basal (p63) and differentiation (K10; Filaggrin) markers after 96 hs of differentiation and treatment with CNO (+CNO) or vehicle (DMSO, −CNO). (g), (h) and (i) Representative quantification of experiments as detailed in (f) (n=25 fields; ***p< 0.001; ****p< 0.0001; t test). Scale bar: 100 μm. (j) Western blot analysis showing the expression of the indicated proteins after stimulation with 1 μM CNO for the specified times. (k) Western blot analysis of the indicated proteins after stimulation with 1 μM CNO or vehicle for 5 min in the presence of 0.1 μg/ml of pertussis toxin (PTX). In (c), (d), (g), (h) and (i) mean ± SD is shown. In IF images inserts show magnification of highlighted area.

We generated N/TERT2G keratinocytes stably expressing hM4Di by lentiviral transduction (Figure 1a). N/TERT2G cells are diploid human keratinocytes immortalized by transduction with human telomerase reverse transcriptase (TERT) and show epidermal differentiation patterns similar to human primary keratinocytes (Dickson et al., 2000, Smits et al., 2017). This model allowed us to have consistent and stable expression of hM4Di, without the inherent variability of independent primary cell isolation or the need for repeated transductions to express the receptor.

Treatment of N/TERT2G hM4Di cells with CNO resulted in a marked increase in proliferation (Figure 1b and c) and reduction in the mRNA levels of differentiation markers, including keratin 1 (KRT1) and transglutaminase 3 (TGM3) (Figure 1d). We also observed increase protein levels of the basal marker p63 and reduced levels of the differentiation markers keratin 10 (K10), filaggrin, keratin 1 (K1) and loricrin (Figure 1e–i and Figure S1B). Interestingly, activation of hM4Di induced a marked increase in proliferative markers, including phospho-ERK and phospho-AKT, as well as phospho-JNK (Figure 1j). hM4Di also induced low levels of YAP1 phosphorylation, which are correlated with increased activation of this co-transcriptional activator (Figure 1j). Induction of ERK and AKT activation was dependent on Gαi as showed by blockage of phosphorylation of these proteins by the Gαi-inhibitor pertussis toxin (PTX) (Figure 1k). Our results indicate that activation of Gαi by GPCRs in keratinocytes triggers MAPK signaling, increasing proliferation and reducing the expression of terminal differentiation markers.

Gαi activation in 3D organotypic human skin cultures induces a hyperplasia phenotype

To study GPCR-Gαi activation in a more physiological context, we next generated 3D organotypic cultures of N/TERT2G hM4Di human keratinocytes. Treatment with CNO in this condition resulted in thickening of epidermal cultures (Figure 2a and b), probably due to reduced differentiation and increased cell proliferation. Indeed, GPCR-Gαi activation lead to decreased expression of the differentiation marker K10 (Figure 2c) and staining with the proliferating cell nuclear antigen (PCNA) revealed that CNO increased proliferation of basal cells (Figure 2d and e). As a control, CNO treatment of keratinocytes not expressing hM4Di had no effect on organotypic culture formation (Figure S2a). The increase in thickness of the organotypic cultures is dependent on Gαi activity, as treatment with PTX restored epidermal thickness of CNO treated cells to those of control cells (Figure 2f and g). To confirm that Gαi activation is involved in the hyperplasia phenotype, we generated N/TERT2G cells expressing a tetracycline-inducible constitutively active mutant of Gαi with an internal EE-epitope tag (tet-Gαi2-Q205L). These cells expressed active Gαi upon doxycycline addition to the culture media (Figure S2b). Organotypic cultures generated with N/TERT2G tet-Gαi2-Q205L showed an increase in thickness upon induction, comparing with the not-induced counterpart (Figure S2c–d). Overall, our data confirms that GTP-bound Gαi is involved in keratinocyte proliferation and differentiation and suggests that Gαi-coupled GPCRs and their ligands could be directly involved in modulating skin hyperplasia.

Figure 2: Gαi activation increases proliferation and epidermal thickening in organotypic cultures of human keratinocytes.

Figure 2:

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(a) H&E staining of epidermis reconstruction assay with hM4Di positive N/TERT2G keratinocytes treated (+CNO, 1 μM) or not (-CNO). Scale bar: 60 μm. (b) Quantification of (a) (n=3; **p< 0.01; t test). (c) and (d) IF showing the expression of differentiation and proliferation markers in organotypic epidermis. Scale bar: 20 μm. (e) Quantification of (d) (n=17–19; *p<0.05; t test). (f) Epidermis reconstruction assay with hM4Di positive N/TERT2G cells treated or not with 1 μM CNO and 0.1 μg/ml pertussis toxin (PTX). Scale bar: 60 μm. (g) Quantification of (f) (n=2; *p<0.05; **p< 0.01; one-way ANOVA followed by t test). In (b), (e) and (g) mean ± SD is shown.

Gαi activation in basal/progenitor epidermal cells induces skin hyperplasia in mice

The study of GPCR signaling is usually complicated by receptors and ligands being expressed in the same cell, and the level of complexity increases when these studies are translated into animal models, where the dissection of the signaling events between different cell types is cumbersome. To circumvent some of these difficulties, we translated our approach in cells to a mouse model by employing tetracycline-inducible hM4Di mice (TRE-hM4Di) (Alexander et al., 2009). Crossing TRE-hM4Di with mice expressing rtTA under the control of a keratin 5 promoter (K5-rtTA) (Vitale-Cross et al., 2004) allowed us to target the expression of hM4Di to the basal/stem cell compartment of the skin (Figure 3a). Mice treated with doxycycline chow showed expression of the receptor at the cell membrane of basal cells in the interfollicular epidermis and hair follicles (Figure 3b and c).

Figure 3: Gαi-coupled GPCR activation in basal cells induces skin hyperplasia in mice.

Figure 3:

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(a) Schematic representation of the animal model used to express hM4Di in the basal compartment of the skin. (b) and (c) IF of tail skin whole mounts showing the expression of HA-tagged hM4Di and basal marker keratin 15 (K15) after induction with doxycycline. Scale bars: 100 μm. (d) Schematic representation of the timeline of doxycycline (Dox) and CNO treatment. (e) Histological analysis of hM4Di positive (hM4Di) and negative (Ctrl) mice after 4 weeks treatment with 2 mg/kg CNO. Scale bar: 100 μm. (f) and (g) Quantification of skin thickness following the indicated time under CNO treatment. Dots represent the mean of epidermis thickness in an individual mouse. In (f) median of the results of control and hM4Di mice is represented as black or red dotted lines, respectively. ((f) n=14 Ctrl mice, n=13 hM4Di mice; ****p< 0.0001; t test; (g) n=3 Ctrl mice and 4 hM4Di mice in week 1, n=4 Ctrl mice and 4 hM4Di mice in week 2, n=4 Ctrl mice and 4 hM4Di mice in week 3, n=4 Ctrl mice and 5 hM4Di mice in week 12; ***p< 0.001; ****p< 0.0001; t test).

We next induced activation GPCR-Gαi signaling in basal keratinocytes by treating mice with CNO by daily intraperitoneal (IP) injection (2mg/kg) (Figure 3d). hM4Di activation resulted in skin hyperplasia after 3 to 4 weeks of treatment (Figure 3e–g). This effect was somehow transient, as hM4Di mice in an extended treatment group (3 months) showed increased but not significant differences in skin thickness compared to control mice (Figure 3g).

hM4Di activation at 4 weeks resulted in an expansion of the K5-positive basal cell compartment and increased proliferation of basal cells, as assessed by PCNA staining, as well as an increased number of p63 positive cells (Figure 4a–c and Figure S3a–b). Interestingly, the activation of hM4Di in vivo also resulted in an increase in the number of differentiated cells labelled by K10 and loricrin (Figure 4a and Figure S2g), probably due to the overall increase in the number of epithelial cells and hM4Di expression being restricted to the basal K5 compartment. Based on previous results showing that Gαs controls keratinocyte proliferation and differentiation by limiting GLI and YAP1 signaling (Iglesias-Bartolome et al., 2015), we hypothesized that activation of Gαi by GPCRs in keratinocytes would oppose Gαs and lead to the activation of these stem cell regulatory proteins. Staining of skin from mice at 4 weeks of treatment showed a marked increase in the nuclear localization of YAP1, suggesting activation of this pathway (Figure 4d and e). Surprisingly, even though Gαi has been proposed to activate hedgehog signaling (Kong et al., 2019, Ogden et al., 2008, Pusapati et al., 2018, Villanueva et al., 2015), we could not detect an increase in protein levels of GLI following hM4Di activation (Figure 4f, basal cell carcinoma following active-smoothen SmoM2 expression is showed as a positive control for GLI staining). To corroborate this finding, we tracked Gli1 positive cells by crossing TRE-hM4Di mice with mice carrying a LacZ gene in the endogenous Gli1 locus (Glilz) (Bai et al., 2002) (Figure 4g). We did not find differences on hedgehog signaling activation between control mice or TRE-hM4Di treated with CNO for 4 weeks (Figure 4h).

Figure 4: Targeted GPCR-Gαi activation causes skin hyperplasia.

Figure 4:

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(a) IF of skin cross sections of hM4Di positive (hM4Di) or negative (Ctrl) mice showing differentiation (K10) and basal/proliferation (K5; PCNA) markers after 4 weeks treatment with CNO. Scale bar: 100 μm. (b) IF of tail skin whole mounts showing expression of the proliferation marker PCNA after 4 weeks treatment with CNO. Scale bar: 50 μm. (c) Quantification of (b) (n=16 to 24 areas; **p< 0.01; t test). (d) IF of tail skin whole mounts showing YAP1 expression in mice after 4 weeks treatment with CNO. Scale bars: 50 μm. (e) Representative quantification (d) (n=15 areas; *p< 0.05; t test). In (c) and (e) mean ± SD is shown. (f) IF of tail skin whole mounts showing the lack of expression of Gli1. The first panel shows skin from a BCC lesion as a positive control for Gli1 staining. Scale bars: 50 μm. (g) Schematic representation of the animal model used to track Gli1 expression. (h) βGal staining for skin cross sections of control (Ctrl)-Glilz and hM4Di-Glilz mice after 4 weeks of CNO treatment. Scale bar: 100 μm. (i) Expression levels of Gnas and Gnai genes in the mouse skin compartments interfollicular epidermis (Epi) and hair follicle outer root sheath (ORS), matrix (Mx), transit amplifying progenitors (TAC), and stem cell precursors (HF-SC). Expression levels expressed as fragments per kilobase of transcript per million mapped reads (FPKM) were obtained from (Rezza et al., 2016). In (a) and (b) inserts show magnification of highlighted area. In (d) inserts show the nuclei. In (c), (e), and (i) mean ± SD is shown.

Our results show that GPCR-Gαi activation in basal keratinocytes causes skin hyperplasia but does not phenocopy the GLI activation and basal cell carcinoma formation observed following epidermal Gαs knockout (Iglesias-Bartolome et al., 2015). This suggests that both heterotrimeric G-proteins might act on different pathways or subsets of cells. Analysis of an available dataset of gene expression among skin cell compartments in mouse skin (Rezza et al., 2016) indicates that levels of each Gnas, Gnai1, Gnai2 and Gnai3 mRNAs (coding for Gαs, Gαi1, Gαi2 and Gαi3 respectively) show only slight differences between keratinocyte skin cell compartments, particularly between interfollicular epidermal cells and hair follicle stem cells (Figure 4i). However, the expression levels for Gnas are more abundant across compartments compared with Gnai genes, indicating the possibility that Gαs and Gαi effects could be related to differential expression levels of these heterotrimeric G proteins.

DISCUSSION

In this study we employ a chemogenetic approach to demonstrate that Gαi-coupled GPCRs can regulate keratinocyte proliferation and differentiation, both in human and mouse skin. By targeting expression of the hM4Di DREADD receptor we were able to study the effects of specific GPCR activation in keratinocytes, avoiding confounding ligand-receptor autocrine and paracrine loops. Our results indicate that ligands that impinge on endogenous Gαi-coupled receptors can regulate skin homeostasis and potentially contribute to skin pathologies. Indeed, ligands for Gαi-coupled GPCRs can alter epidermal homeostasis: increase in the levels of S1P, which binds and activates Gαi-coupled sphingosine-1-phosphate receptors (S1PR), leads to skin hyperplasia (Allende et al., 2013); LPA induces keratinocyte proliferation, skin hyperplasia and accelerated wound healing (Balazs et al., 2001, Piazza et al., 1995) by binding to lysophosphatidic acid receptors (LPAR), some of which can activate Gαi; elevated levels of LTB4 during allergic skin inflammation lead to epidermal hyperplasia through the Gαi-coupled LTB4 receptor (LTB4R, also known as BLT1) (Oyoshi et al., 2012); and activation of F2R Like Trypsin Receptor 1 (F2RL1 also known as PAR-2) has been involved in skin hyperplasia and squamous cell carcinogenesis by activating Gαi (Sales et al., 2015). While in most cases it is not clear the exact cell population responsible for the phenotypes induced by GPCRs and their ligands, our results suggest that the direct action of inflammatory mediators and other ligands in keratinocytes could mediate some of the observed epithelial effects.

We have previously demonstrated that Gnas deletion in the mouse skin leads to a blockage on basal stem and progenitor cell differentiation, causing basal cell carcinoma (Iglesias-Bartolome et al., 2015). Here we show that increased Gαi activity in keratinocytes, which opposes Gαs by reducing cAMP levels, causes skin hyperplasia but does not phenocopy epidermal Gαs knockout. Expression levels of Gnas are consistently higher in different skin keratinocyte compartments when compared to Gnai genes, suggesting that phenotypic differences might be a result of differential expression of these molecules. The increased expression of Gαs could have a dominant effect on GLI-inhibition over Gαi activation of this pathway. Other explanations are possible, including different subcellular localization in cAMP-regulation of hedgehog signaling downstream of Gαs and Gαi in vivo.

Lastly, our experimental results show that GPCR-Gαi signaling in keratinocytes triggers the activation of MAPK, AKT and YAP1, which potentially contribute to the observed increase in keratinocyte proliferation and reduced differentiation. Indeed, GPCR signaling triggers multiple mitogenic pathways (Dorsam and Gutkind, 2007), including ERK and JNK, that are essential for epithelial proliferation under normal and pathological conditions (Scholl et al., 2007, Weston et al., 2004). GPCR-Gαi signaling also results in activation of YAP1 by dephosphorylation and nuclear translocation. YAP1 is a co-transcriptional activator central for the proliferation of epithelial progenitors and cancer formation in the skin (Schlegelmilch et al., 2011, Walko et al., 2017). It is important to note that additional mitogenic contributions from GPCR signaling independent of Gαi-cAMP signaling and dependent on the activation of PI3K and MAPK through βγ subunits are possible (Crespo et al., 1994).

Since GPCRs and their downstream effectors are critical components in tissue homeostasis and human malignancies (Bailey et al., 2018, O’Hayre et al., 2013), genetic and chemogenetic approaches that help us understand how GPCR signaling interfaces with developmental pathways in normal skin physiology and disease could aid in the design of novel regenerative approaches and intervention strategies for degenerative and neoplastic conditions.

MATERIALS & METHODS

Detailed information on primers, antibodies and quantification methods is available in Supplementary Text file.

DNA constructs:

hM4Di was cloned from the HA-tagged DREADD sequence (Addgene plasmid #50471, gift from Bryan Roth). Gαi2-Q205L was purchased from the cDNA Resource Center, www.cdna.org, catalog #GNAI20EIC0. Lentivirus were produced by transfecting Lenti-X™ 293T cells with the transfer and packaging plasmids.

Mice:

All animal studies were carried out according to NIH-Intramural Animal Care and Use Committee (ACUC) approved protocols, in compliance with the Guide for the Care and Use of Laboratory Animals. FVB/N K5-rtTA mice have been previously described (Vitale-Cross et al., 2004). TRE-hM4Di mice (Alexander et al., 2009), Glilz reporter mice (Bai et al., 2002) and SmoM2 (Jeong et al., 2004) mice were obtained from The Jackson Laboratory (Stock Number: 024114; 008211 and 005130 respectively). Both male and female mice were used in the studies. Experiments were conducted using littermate controls that did not express hM4Di and all control and hM4Di mice were treated with doxycycline and CNO. Doxycycline was administered in the food grain-based pellets (Bio-Serv) at 6g kg-1. CNO (Tocris, catalog no. 4936) was resuspended in DMSO at a concentration of 50 mg/ml. The stock solution was diluted in PBS before injection in mice at a final concentration of 2mg/kg. Treatment was started between weeks 6 to 10 after birth.

Cell culture, transfections and lentiviral transductions:

All cells were cultured at 37°C in the presence of 5% CO2. Lenti-X™ 293T and NIH3T3 cells were obtained from Takara Bio and AddexBio, respectively, and cultured in DMEM (Sigma-Aldrich Inc) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich Inc) and antibiotic/antimycotic solution (Sigma-Aldrich Inc). N/TERT2G keratinocyte cell line (Dickson et al., 2000, Smits et al., 2017) was provided by Dr. Ellen H. van den Bogaard (Radboud University Medical Center, Nijmegen, The Netherlands) and Dr. James Rheinwald (Brigham and Women’s Hospital, Boston, MA, USA). N/TERT2G cells were tested for mycoplasma by PCR and validated using small tandem repeat (STR) profiling. N/TERT2G keratinocytes were cultured in EpiLife media (Life Technologies) with Human Keratinocyte Growth Supplement (HKGS, Life Technologies). To trigger N/TERT2G differentiation, 100% confluent cells were incubated with CaCl2 at a final concentration of 1.5 mM for 96 hs. To trigger expression of Gαi2-Q205L, stable cells were incubated overnight with 1 μg/ml of doxycycline to induce the expression of the constitutively active mutant. The expression of the protein was confirmed by Western blot.

Immunoblot Analysis:

Western blot assays were performed as described previously (Iglesias-Bartolome et al., 2012, Vaque et al., 2013). For treatment with CNO, cells were starved overnight and stimulated with 1 μM CNO for the indicated times. For experiments performed in the presence of PTX (Tocris; catalog no. 3097), cells were incubated with 0.1 μg/ml of the toxin for 4 hs before starting the experiment. Bands were detected using a ChemiDoc™ Imaging System (Bio-Rad) with Clarity™ Western ECL Blotting Substrates (Bio-Rad) according to the manufacturer’s instructions.

Immunofluorescence and Immunohistochemistry:

For immunofluorescence cells were fixed with 3.2% paraformaldehyde in PBS, permeabilized with 0.05% Triton X-100 and 200 mM glycine in PBS and blocked with BSA 3% in PBS. Cells were incubated with the primary antibodies overnight at 4°C, followed by 2 hs incubation with the secondary antibodies. Immunofluorescence analysis of mouse skin was performed on tissue sections embedded in paraffin or whole mounts as indicated. Sections, tail skin whole mounts and βGal stainings were performed as previously described (Iglesias-Bartolome et al., 2015). For histological analysis, tissues were embedded in paraffin; 3-μm sections were obtained and stained with H&E. H&E slides were scanned at 40× using an Aperio CS Scanscope (Aperio) and analyzed with Aperio ImageScope software. Each immunostaining was repeated at least in 3 independent mice or 3 independent experiments and several fields were reviewed.

Cell proliferation:

Cell proliferation was evaluated using 4 hs incorporation of EdU followed by labeling with the Click-IT EdU Imaging Kit (Invitrogen) according to the manufacturer’s instructions. The proportion of positive cells was determined as explained above.

Organotypic cultures:

HEK or N/TERT2G cells were used to perform human epidermis reconstruction experiments based on (De Vuyst et al., 2014, Smits et al., 2017) with some modifications. Briefly, 1×105 cells were plated in a 24 well plate insert (Corning; catalog no. CLE3470–48EA) in EpiLife media supplemented with 50 μg/ml L-ascorbic acid (Sigma; catalog no. A4403) and 10 ng/ml keratinocyte growth factor (Sigma; catalog no. K1757). After 48 hs, cells were exposed to the air, and the media outside the insert was replaced with the supplemented media containing 1.5 mM CaCl2. CNO (1 μM), PTX (0.1 μg/ml), Doxycycline (1 μg/ml) or vehicle were added in the respective concentrations. The media was renewed every 2 days for 11 days, then inserts containing the fully differentiated epidermis were fixed overnight in Z-Fix, embedded in paraffin, and prepared for histological analysis.

Statistical analysis:

Statistical analyses, variation estimation and validation of test assumptions were carried out using the Prism 5 statistical analysis program (GraphPad). Asterisks denote statistical significance (non-significant or NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). All data are reported as mean ± standard deviation (SD).

Data availability statement:

Gnas and Gnai gene expression in mouse skin were analyzed from GSE77197 hosted at https://www.ncbi.nlm.nih.gov/geo/. Any additional data are available from the corresponding author upon reasonable request.

Supplementary Material

1
Fig 1. Supplementary Figure S1: hM4Di regulation of intracellular cAMP and protein expression of differentiation markers.

(a) Quantification of cAMP concentration using a fluorescent cAMP detector after the addition of 1 μM CNO (+CNO) or vehicle (DMSO, −CNO) to NIH3T3 cells expressing hM4Di. Results are normalized to the initial fluorescence level, shown as a red dotted line. Black arrow represents the addition of CNO. Reporter fluorescence intensity increase indicates cAMP levels decrease (Tewson et al., 2018). Mean ± SD is shown for each time point. (b) Western blot analysis of expression of basal (K5; p63) and differentiation (K10; K1; loricrin) markers in differentiated hM4Di positive cells treated (+CNO) or not (-CNO) with CNO.

Fig 2. Supplementary Figure S2: Effect of constitutive activation of Gαi in organotypic cultures.

(a) Epidermis reconstruction assay of normal human keratinocytes (not expressing hM4Di) treated (+CNO) or not (-CNO) with 1 μM CNO. Scale bar: 60 μm. (b) Western blot showing the expression of EE-tagged Gαi2-Q205L in stable N/TERT2G cells incubated (+Dox) or not (-Dox) with doxycycline (1 μg/ml). (c) Epidermis reconstruction assay of inducible N/TERT2G Gαi2-Q205L, treated (+Dox) or not (-Dox) with 1 μg/ml doxycycline. Scale bar: 50 μm. (d) Quantification of (c) (n=3; **p<0.01; one-way ANOVA followed by t test). Mean ± SD is shown.

Fig 3. Supplementary Figure S3: Expression of basal and differentiation markers in hM4Di mouse skin.

(a) and (b) IF of skin cross sections of hM4Di positive (hM4Di) or negative (Ctrl) mice showing differentiation (Loricrin) and basal (K5; p63) markers after 4 weeks treatment with CNO. Scale bar: 20 μm.

ACKNOWLEDGMENTS

This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (ZIA BC 011764 and ZIA BC 011763).

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

REFERENCES

  1. Alexander GM, Rogan SC, Abbas AI, Armbruster BN, Pei Y, Allen JA, et al. Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 2009;63(1):27–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allende ML, Sipe LM, Tuymetova G, Wilson-Henjum KL, Chen W, Proia RL. Sphingosine-1-phosphate phosphatase 1 regulates keratinocyte differentiation and epidermal homeostasis. The Journal of biological chemistry 2013;288(25):18381–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bai CB, Auerbach W, Lee JS, Stephen D, Joyner AL. Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway. Development 2002;129(20):4753–61. [DOI] [PubMed] [Google Scholar]
  4. Bailey MH, Tokheim C, Porta-Pardo E, Sengupta S, Bertrand D, Weerasinghe A, et al. Comprehensive Characterization of Cancer Driver Genes and Mutations. Cell 2018;173(2):371–85 e18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Balazs L, Okolicany J, Ferrebee M, Tolley B, Tigyi G. Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo. Am J Physiol Regul Integr Comp Physiol 2001;280(2):R466–72. [DOI] [PubMed] [Google Scholar]
  6. Belokhvostova D, Berzanskyte I, Cujba AM, Jowett G, Marshall L, Prueller J, et al. Homeostasis, regeneration and tumour formation in the mammalian epidermis. Int J Dev Biol 2018;62(6-7-8):571–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Callihan P, Mumaw J, Machacek DW, Stice SL, Hooks SB. Regulation of stem cell pluripotency and differentiation by G protein coupled receptors. Pharmacol Ther 2011;129(3):290–306. [DOI] [PubMed] [Google Scholar]
  8. Crespo P, Xu N, Simonds WF, Gutkind JS. Ras-dependent activation of MAP kinase pathway mediated by G-protein beta gamma subunits. Nature 1994;369(6479):418–20. [DOI] [PubMed] [Google Scholar]
  9. De Vuyst E, Charlier C, Giltaire S, De Glas V, de Rouvroit CL, Poumay Y. Reconstruction of normal and pathological human epidermis on polycarbonate filter. Methods in molecular biology 2014;1195:191–201. [DOI] [PubMed] [Google Scholar]
  10. Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, et al. Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 2000;20(4):1436–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dorsam RT, Gutkind JS. G-protein-coupled receptors and cancer. Nature reviews Cancer 2007;7(2):79–94. [DOI] [PubMed] [Google Scholar]
  12. Iglesias-Bartolome R, Gutkind JS. Keeping the epidermal stem cell niche in shape. Cell stem cell 2010;7(2):143–5. [DOI] [PubMed] [Google Scholar]
  13. Iglesias-Bartolome R, Patel V, Cotrim A, Leelahavanichkul K, Molinolo AA, Mitchell JB, et al. mTOR inhibition prevents epithelial stem cell senescence and protects from radiation-induced mucositis. Cell stem cell 2012;11(3):401–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iglesias-Bartolome R, Torres D, Marone R, Feng X, Martin D, Simaan M, et al. Inactivation of a Galpha(s)-PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis. Nature cell biology 2015;17(6):793–803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jeong J, Mao J, Tenzen T, Kottmann AH, McMahon AP. Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. Gene Dev 2004;18(8):937–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kobayashi NR, Hawes SM, Crook JM, Pebay A. G-protein coupled receptors in stem cell self-renewal and differentiation. Stem cell reviews 2010;6(3):351–66. [DOI] [PubMed] [Google Scholar]
  17. Kong JH, Siebold C, Rohatgi R. Biochemical mechanisms of vertebrate hedgehog signaling. Development 2019;146(10). [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mo JS, Park HW, Guan KL. The Hippo signaling pathway in stem cell biology and cancer. EMBO reports 2014;15(6):642–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. O’Hayre M, Vazquez-Prado J, Kufareva I, Stawiski EW, Handel TM, Seshagiri S, et al. The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer. Nature reviews Cancer 2013;13(6):412–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ogden SK, Fei DL, Schilling NS, Ahmed YF, Hwa J, Robbins DJ. G protein Galphai functions immediately downstream of Smoothened in Hedgehog signalling. Nature 2008;456(7224):967–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Oyoshi MK, He R, Li Y, Mondal S, Yoon J, Afshar R, et al. Leukotriene B4-driven neutrophil recruitment to the skin is essential for allergic skin inflammation. Immunity 2012;37(4):747–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Piazza GA, Ritter JL, Baracka CA. Lysophosphatidic acid induction of transforming growth factors alpha and beta: modulation of proliferation and differentiation in cultured human keratinocytes and mouse skin. Exp Cell Res 1995;216(1):51–64. [DOI] [PubMed] [Google Scholar]
  23. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nature reviews Molecular cell biology 2002;3(9):639–50. [DOI] [PubMed] [Google Scholar]
  24. Pusapati GV, Kong JH, Patel BB, Krishnan A, Sagner A, Kinnebrew M, et al. CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog. Developmental cell 2018;44(2):271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rezza A, Wang Z, Sennett R, Qiao W, Wang D, Heitman N, et al. Signaling Networks among Stem Cell Precursors, Transit-Amplifying Progenitors, and their Niche in Developing Hair Follicles. Cell reports 2016;14(12):3001–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sales KU, Friis S, Konkel JE, Godiksen S, Hatakeyama M, Hansen KK, et al. Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis. Oncogene 2015;34(3):346–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Santos R, Ursu O, Gaulton A, Bento AP, Donadi RS, Bologa CG, et al. A comprehensive map of molecular drug targets. Nat Rev Drug Discov 2017;16(1):19–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schlegelmilch K, Mohseni M, Kirak O, Pruszak J, Rodriguez JR, Zhou D, et al. Yap1 acts downstream of alpha-catenin to control epidermal proliferation. Cell 2011;144(5):782–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scholl FA, Dumesic PA, Barragan DI, Harada K, Bissonauth V, Charron J, et al. Mek1/2 MAPK kinases are essential for Mammalian development, homeostasis, and Raf-induced hyperplasia. Developmental cell 2007;12(4):615–29. [DOI] [PubMed] [Google Scholar]
  30. Smits JPH, Niehues H, Rikken G, van Vlijmen-Willems I, van de Zande G, Zeeuwen P, et al. Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models. Scientific reports 2017;7(1):11838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tewson P, Martinka S, Shaner N, Berlot C, Quinn AM, Hughes T. Assay for Detecting Galphai-Mediated Decreases in cAMP in Living Cells. SLAS Discov 2018;23(9):898–906. [DOI] [PubMed] [Google Scholar]
  32. Urban DJ, Roth BL. DREADDs (designer receptors exclusively activated by designer drugs): chemogenetic tools with therapeutic utility. Annu Rev Pharmacol Toxicol 2015;55:399–417. [DOI] [PubMed] [Google Scholar]
  33. Vaque JP, Dorsam RT, Feng X, Iglesias-Bartolome R, Forsthoefel DJ, Chen Q, et al. A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors. Molecular cell 2013;49(1):94–108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Villanueva H, Visbal AP, Obeid NF, Ta AQ, Faruki AA, Wu MF, et al. An essential role for Galpha(i2) in Smoothened-stimulated epithelial cell proliferation in the mammary gland. Science signaling 2015;8(394):ra92. [DOI] [PubMed] [Google Scholar]
  35. Vitale-Cross L, Amornphimoltham P, Fisher G, Molinolo AA, Gutkind JS. Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis. Cancer research 2004;64(24):8804–7. [DOI] [PubMed] [Google Scholar]
  36. Walko G, Woodhouse S, Pisco AO, Rognoni E, Liakath-Ali K, Lichtenberger BM, et al. A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells. Nat Commun 2017;8:14744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weston CR, Wong A, Hall JP, Goad ME, Flavell RA, Davis RJ. The c-Jun NH2-terminal kinase is essential for epidermal growth factor expression during epidermal morphogenesis. Proceedings of the National Academy of Sciences of the United States of America 2004;101(39):14114–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Williams SE, Beronja S, Pasolli HA, Fuchs E. Asymmetric cell divisions promote Notch-dependent epidermal differentiation. Nature 2011;470(7334):353–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Williams SE, Ratliff LA, Postiglione MP, Knoblich JA, Fuchs E. Par3-mInsc and Galphai3 cooperate to promote oriented epidermal cell divisions through LGN. Nature cell biology 2014;16(8):758–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yu FX, Zhao B, Panupinthu N, Jewell JL, Lian I, Wang LH, et al. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell 2012;150(4):780–91. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1542207-supplement-1.docx (48.3KB, docx)
Fig 1. Supplementary Figure S1: hM4Di regulation of intracellular cAMP and protein expression of differentiation markers.

(a) Quantification of cAMP concentration using a fluorescent cAMP detector after the addition of 1 μM CNO (+CNO) or vehicle (DMSO, −CNO) to NIH3T3 cells expressing hM4Di. Results are normalized to the initial fluorescence level, shown as a red dotted line. Black arrow represents the addition of CNO. Reporter fluorescence intensity increase indicates cAMP levels decrease (Tewson et al., 2018). Mean ± SD is shown for each time point. (b) Western blot analysis of expression of basal (K5; p63) and differentiation (K10; K1; loricrin) markers in differentiated hM4Di positive cells treated (+CNO) or not (-CNO) with CNO.

NIHMS1542207-supplement-Fig_1.tif (3.4MB, tif)
Fig 2. Supplementary Figure S2: Effect of constitutive activation of Gαi in organotypic cultures.

(a) Epidermis reconstruction assay of normal human keratinocytes (not expressing hM4Di) treated (+CNO) or not (-CNO) with 1 μM CNO. Scale bar: 60 μm. (b) Western blot showing the expression of EE-tagged Gαi2-Q205L in stable N/TERT2G cells incubated (+Dox) or not (-Dox) with doxycycline (1 μg/ml). (c) Epidermis reconstruction assay of inducible N/TERT2G Gαi2-Q205L, treated (+Dox) or not (-Dox) with 1 μg/ml doxycycline. Scale bar: 50 μm. (d) Quantification of (c) (n=3; **p<0.01; one-way ANOVA followed by t test). Mean ± SD is shown.

NIHMS1542207-supplement-Fig_2.tif (2.5MB, tif)
Fig 3. Supplementary Figure S3: Expression of basal and differentiation markers in hM4Di mouse skin.

(a) and (b) IF of skin cross sections of hM4Di positive (hM4Di) or negative (Ctrl) mice showing differentiation (Loricrin) and basal (K5; p63) markers after 4 weeks treatment with CNO. Scale bar: 20 μm.

NIHMS1542207-supplement-Fig_3.tif (4.7MB, tif)

Data Availability Statement

Gnas and Gnai gene expression in mouse skin were analyzed from GSE77197 hosted at https://www.ncbi.nlm.nih.gov/geo/. Any additional data are available from the corresponding author upon reasonable request.

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