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. Author manuscript; available in PMC: 2025 Jun 1.
Published in final edited form as: J Invest Dermatol. 2023 Dec 14;144(6):1311–1321.e7. doi: 10.1016/j.jid.2023.12.002

GPCR screening reveals that the metabolite receptor HCAR3 regulates epithelial proliferation, migration, and cellular respiration

M Pilar Pedro 1, Katherine Lund 1, Sun Woo Sophie Kang 2, Ting Chen 1, Christina H Stuelten 1, Natalie Porat-Shliom 2, Ramiro Iglesias-Bartolome 1,*
PMCID: PMC11116076  NIHMSID: NIHMS1952988  PMID: 38103827

Abstract

Epithelial cells in the skin and other tissues rely on signals from their environment to maintain homeostasis and respond to injury, and G protein-coupled receptors (GPCRs) play a critical role in this communication. A better understanding of the GPCRs expressed in epithelial cells will contribute to understanding the relationship between cells and their niche and could lead to developing new therapies to modulate cell fate. This study used human primary keratinocytes as a model to investigate the specific GPCRs regulating epithelial cell proliferation and differentiation. We identified three key receptors, hydroxycarboxylic acid-receptor 3 (HCAR3), leukotriene B4-receptor 1 (LTB4R), and G Protein-Coupled Receptor 137 (GPR137) and found that knockdown of these receptors led to changes in numerous gene networks that are important for maintaining cell identity and promoting proliferation while inhibiting differentiation. Our study also revealed that the metabolite receptor HCAR3 regulates keratinocyte migration and cellular metabolism. Knockdown of HCAR3 led to reduced keratinocyte migration and respiration, which could be attributed to altered metabolite use and aberrant mitochondrial morphology caused by the absence of the receptor. This study contributes to understanding the complex interplay between GPCR signaling and epithelial cell fate decisions.

Introduction

The skin relies on self-renewing keratinocyte stem cells to replenish lost cells and heal wounds, making it a valuable system for studying cell fate decisions (Belokhvostova et al., 2018, Blanpain and Fuchs, 2009, Watt, 2014). The fate of basal keratinocytes includes either proliferation, to generate stem cells and transient-amplifying cells, or differentiation, which maintains the multilayer epidermis and its barrier function. The balance between proliferation and differentiation is crucial for proper tissue homeostasis, and its dysregulation can lead to aging-related disorders, impaired tissue regeneration, and cancer (Hanahan and Weinberg, 2011, Iglesias-Bartolome and Gutkind, 2011, Lopez-Otin et al., 2013).

Keratinocytes must adequately sense their microenvironment to coordinate cell fate decisions, respond to stress, and restore homeostasis. G-protein-coupled receptors (GPCRs), the largest family of membrane signal transducers (Pierce, Premont and Lefkowitz, 2002), are capable of sensing multiple extracellular molecules, including hormones, inflammatory mediators, nutrients, and metabolites, and can regulate cell fate in stem cells (Callihan et al., 2011, Kobayashi et al., 2010). Understanding the GPCR-regulated signaling pathways in skin biology will shed light on the mechanisms stem cells use to sense and adapt to environmental changes. Moreover, given that more than one-third of all therapeutic drugs target GPCRs (Santos et al., 2017), identifying receptors involved in epithelial cell fate regulation can facilitate the development of pharmacological interventions to increase tissue regenerative capacity and modulate pathological conditions.

GPCRs relay their signaling through heterotrimeric G protein-dependent and independent pathways and are categorized into five classes: A (rhodopsin), B (secretin), C (glutamate), Frizzled, and Adhesion. Analysis of gene expression in mouse epidermis has shown that multiple GPCRs are expressed in keratinocytes, and the function of some of these receptors in the regulation of skin biology is well known (Pedro, Lund and Iglesias-Bartolome, 2020). However, the functions of many GPCRs remain unclear due to their genetic and signaling redundancy and broad expression in multiple cell types. In addition, studies on GPCR pathways usually employ mouse models, highlighting the need for further investigation into the specific effects of GPCRs in human cells.

This study aimed to advance our understanding of epidermal cell fate regulation by GPCRs by characterizing the endogenous receptors expressed in human primary keratinocytes. Our findings reveal that hydroxycarboxylic acid-receptor 3 (HCAR3), leukotriene B4-receptor 1 (LTB4R), G Protein-Coupled Receptor 153 (GPR153), and G Protein-Coupled Receptor 137 (GPR137), play essential roles in modulating human keratinocyte proliferation and differentiation through the regulation of diverse gene expression networks. Overexpression of HCAR3, LTB4R, and GPR137, but not GPR153, resulted in aberrant development and differentiation of keratinocytes in 3D organotypic cultures. We also discovered that HCAR3, an understudied receptor exclusive to higher primates, is involved in keratinocyte migration and metabolism. Remarkably, HCAR3 regulates oxidative phosphorylation and fatty acid oxidation and is required to sustain proper mitochondrial energy output in keratinocytes. Our study provides a valuable resource for characterizing GPCRs in epithelial cells and sheds light on the potential role of cellular metabolism in keratinocyte proliferation, differentiation, and migration.

Results

Profiling GPCRs in human keratinocytes

To gain a better understanding of the GPCR landscape in keratinocytes, we conducted RNA sequencing (RNAseq) analysis on primary human epidermal keratinocytes (HEK) under non-differentiating and calcium-induced differentiation conditions (Figure 1a). Our analysis revealed high expression levels of several GPCRs known to regulate skin biology (Figure 1b and Table S1). Of note, the β2 adrenergic receptor (ADRB2) has a role in wound closure in mice (Pullar et al., 2012, Sivamani et al., 2014); LTB4R is involved in allergic skin inflammation (Oyoshi et al., 2012); sphingosine-1-phosphate receptors (S1PR) ligand is involved in skin hyperplasia (Allende et al., 2013); the F2R Like Trypsin Receptor 1 (F2RL1, also known as PAR-2) is involved in skin hyperplasia and squamous cell carcinogenesis (Sales et al., 2015); and the lysophosphatidic acid receptors (LPAR) ligand induces keratinocyte proliferation, skin hyperplasia and wound healing (Balazs et al., 2001, Piazza, Ritter and Baracka, 1995).

Figure 1: GPCRs regulating human keratinocyte proliferation.

Figure 1:

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(a) Schematic representation of RNA-seq analysis of human skin keratinocytes (HEK) under basal and differentiated conditions. (b) Circos plot representing the GPCR expression in HEK cells showing class, gene name, Gα-coupling, level of expression (log2 of transcripts per million, Log2TPM), and fold change (FC) over basal/non-differentiated cells. Blue and red squares represent down- (q<0.05, FC≤−1.5) or up- (q<0.05, FC≥1.5) regulation, respectively. GPCRs with expression levels of TPM≥0.5 are shown. (c) qRT-PCR analysis showing the remaining expression of the selected GPCRs in HEK cells 72 h after transfection with the corresponding siRNA for that gene. (d) Representative images of EdU staining of siCTRL and siGPCRs transfected cells. Nuclei staining is shown in white, and EdU-positive cells are colored red. Scale bar: 100 μm. (e) Quantification of EdU-proliferation assay in primary HEK cells, 72hs after siRNA transfection with the indicated siRNAs (Class A top panel, non-Class A bottom panel). Results are normalized to non-targeting siRNA, represented as a black dotted line. Values of ±20% change in proliferation are shown as red dotted lines. n=4-5 independent experiments. (f) Proliferation scores comparing individual versus pool siRNA experiments (calculated as indicated in the methods section). (g) Quantification of EdU-proliferation assays in N/TERT2G cells presented as in (e) (n=5 independent experiments). (h-i) Quantification of EdU-proliferation assays in HEK (h) or N/TERT2G (i) cells grown in defined media and presented as in (e) (n=3 independent experiments). In (e), (g), (h), and (i): no asterisk p>0.05; *p<0.05; **p<0.01; ***p< 0.001; ****p< 0.0001; 2-way ANOVA followed by t-test.

We screened a set of the 24 highest expressed receptors with a pooled-small interference RNA (siRNA) library to identify the GPCRs affecting human keratinocyte proliferation (Figure 1c). Knockdown of LTB4R, HCAR3, GPR153, and GPR137 significantly decreased primary HEK proliferation by more than 20% compared to non-targeting siRNA (Figure 1d and e). The knockdown of LTB4R and GPR137 also reduced the proliferation of differentiated keratinocytes (Figure S1a). Notably, knockdown of the adhesion receptor ADGRG1 (also known as GPR56) and Frizzled Class Receptor 2 (FZD2) significantly increased the proliferation of differentiated HEK (Figure S1a).

To confirm and validate the pooled siRNA results, we used four individual siRNAs to knockdown the identified GPCRs (Figure S1b). Knockdown of LTB4R, HCAR3, GPR153, and GPR137 significantly reduced keratinocyte proliferation, while the efficacy of ADGRG1 and FZD2 siRNAs was inconsistent, suggesting possible off-target effects (Figure 1f and Figure S1c). Moreover, LTB4R, HCAR3, GPR153, and GPR137 knockdown reduced proliferation of immortalized human N/TERT2G keratinocytes (Figure 1g, Figure S1d and e). siRNAs for the GPCRs reduced proliferation in both full media (containing bovine pituitary extract, Figure 1e and g) and defined media (Figure 1h, 1i), indicating that the effects were not dependent on potential GPCR ligands present in the pituitary extract. This result also suggests that the potential ligand activating the GPCRs is produced by the keratinocytes themselves. Analysis of single cell data from human skin (Karlsson et al., 2021) confirmed the expression of LTB4R, HCAR3, GPR153, and GPR137 in basal and differentiated (suprabasal) keratinocytes (Figure S1f). Based on our results, we focused on LTB4R (also known as BLT1), HCAR3 (also known as GPR109B or HCA3), and the orphan receptors GPR153 and GPR137. Both LTB4R and HCAR3 are Gαi coupled receptors, highlighting the significance of this pathway in regulating keratinocyte cell growth (Pedro et al., 2020).

HCAR3, LTB4R, and GPR137 modulate transcriptional networks involved in keratinocyte proliferation and differentiation

To elucidate the mechanisms and signaling pathways responsible for the observed phenotypes, we performed RNAseq in primary HEK transfected with pooled siRNAs for LTB4R, HCAR3, GPR153, GPR137, and non-targeting sequence control. Principal component analysis (PCA) and Pearson correlation of the global RNA levels showed differential clustering of the siGPCRs with respect to control samples (Figure S2a and b). Examination of differentially regulated transcripts (Table S2) revealed that GPCRs affected the expression of numerous genes (Figure 2a). Gene ontology (GO) analysis of biological processes showed that 234 overlapping genes were involved in cell cycle processes (Figure 2b), emphasizing the roles of the receptors in regulating keratinocyte proliferation.

Figure 2: GPCRs control proliferation and differentiation gene networks.

Figure 2:

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(a) Venn diagram showing the number of genes up- and down-regulated (q<0.05, FC≥±1.5) in RNAseq analysis of siGPCRs transfected cells. Common differentially regulated genes for all the siGPCRs are indicated in the middle (234). (b) Graph indicating the gene ontology (GO) biological process terms enriched in common differentially regulated genes. (c) Gene set enrichment analysis showing the normalized enrichment score (NES) for selected GO biological processes in each GPCR dataset. (d) Graph indicating expression fold change (Log2 FC) of genes related to canonical signaling pathways in each of the siGPCRs datasets. (e) IPA functional analysis of the activation state of upstream regulators with the indicated cell functions.

GO with normalized enrichment scores (NES) of biological processes in each dataset indicated that knockdown of LTB4R, HCAR3, and GPR137 activated differentiation and skin development, while downregulated processes related to cell cycle and DNA replication (Figure 1c and Table S3). The knockdown of GPR153 downregulated both keratinocyte differentiation and cell cycle networks (Figure 1c and Table S3). Consistent with these findings, the siRNAs for LTB4R, HCAR3, and GPR137, but not GPR153 upregulated genes associated with all stages of keratinocyte differentiation (Figure S2c). Interestingly, knockdown of the GPCRs showed a stronger effect on the expression of differentiation markers compared with basal markers (Figure S2d). Western blot analysis for the differentiation markers KLF4 and keratin 10 (K10) confirmed the activation of differentiation programs following the knockdown of LTB4R, HCAR3, and GPR137 (Figure S2e). These results indicate that the GPCRs might be affecting the initiation of differentiation more than the expression of basal keratinocyte markers.

Analysis of gene expression changes indicated a specific knockdown for the analyzed GPCRs without significantly affecting related family members (Figure 2d, GPCRs; Table S2). All four GPCRs showed common gene regulation trends on key epidermal pathways (Figure 2d). Knockdown of LTB4R, HCAR3, GPR153, and GPR137 resulted in reduced Hippo signaling targets and differential regulation of NFKB, Wnt, Notch, and Hedgehog genes (Figure 2d). Ingenuity pathway analysis (IPA) of transcriptional regulator networks confirmed that cell cycle-related networks, MYC and YAP1, are particularly affected by the knockdown of these GPCRs (Figure 2e). Cyclic AMP (cAMP)-related gene networks were differentially regulated by LTB4R and HCAR3 (Figure 2e), possibly due to reduced Gαi signaling resulting from the knockdown of these receptors.

Our gene expression analysis confirms the regulation of cell cycle gene networks by LTB4R, HCAR3, GPR153, and GPR137 and reveals numerous pathways affected by their downregulation. To further investigate these GPCRs, we generated doxycycline (Dox) inducible (i) N/TERT2G cells that overexpress LTB4R, HCAR3, GPR153, and GPR137 (iLTB4R, iHCAR3, iGPR153, and iGPR137; Figure S3a). We found that overexpression of all four GPCRs increased AKT phosphorylation, while HCAR3 and GPR137 also activated ERK phosphorylation (Figure 3a). In addition, HCAR3 induced activation of the mTOR target pS6 and reduced pCREB (Figure 3a), a target for cAMP and protein kinase A (PKA).

Figure 3: GPCR overexpression affects proliferation/differentiation balance and skin development.

Figure 3:

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(a) Western blot showing expression of the indicated markers in cells treated (+) or not (−) with doxycycline (Dox) for 96 h to induce expression in inducible (i) GPCR stable cell lines. Relative levels for the phosphorylated bands normalized to the respective total protein and divided by the −Dox control is shown to the right. (b) Western blot showing the expression of keratin 10 (K10) in cells treated as indicated in (a). (c) Hematoxylin & Eosin staining of organotypic assays without (−Dox) or with (+Dox) doxycycline to induce expression of the indicated GPCRs. Scale bar: 60μm. (d) Quantification of organotypic cultures thickness shown in (c). n=3 independent experiments performed in duplicates; one-way ANOVA followed by t-test. (e) Representative images of immunofluorescence experiments showing the expression of keratin 10 (K10) in green, keratin 5 (K5) in red, and nuclei in blue in the organotypic cultures presented in (c). Scale bar: 50 μm.

Confirming the effect of the GPCRs in keratinocyte differentiation, we observed a reduction of K10 in a time-dependent manner upon expression of LTB4R, HCAR3, and GPR137, but not GPR153 (Figure 3b). HCAR3 and GPR137 reduced K10 expression even under differentiating conditions (Figure S3b). In 3D skin-organotypic cultures, we observed abnormal development upon expressing LTB4R, HCAR3, and GPR137, while GPR153 did not have an effect (Figure 3c and d). LTB4R, HCAR3, and GPR137 reduced differentiated cells as labeled by K10 and K6 expression (Figure 3e and Figure S3c), indicating that the reduction in skin thickness upon overexpression of the GPCRs could be due to aberrant differentiation. These findings suggest that GPCR-regulated gene networks play a critical role in human keratinocyte proliferation and differentiation and that their downregulation or overexpression can lead to altered epidermal homeostasis.

HCAR3 regulates keratinocyte migration

Our RNAseq analysis revealed that GPCR knockdown led to differential regulation of gene networks related to keratinocyte migration, particularly in siHCAR3 cells (Figure 2C and Table S3). Given the crucial role of migration in skin wound healing, we conducted scratch-wound assays to examine the effect of LTB4R, HCAR3, GPR153, and GPR137 knockdown in wound closure. Our findings indicate that HCAR3 knockdown had the most profound impact, causing a marked reduction in wound closure in primary and N/TERT2G human keratinocytes (Figure 4a, b, c, and d; Figure S4a, b, and c). siRNA for HCAR3 reduced keratinocyte movement, as measured by accumulated distance travelled by individual cells during the scratch-wound assay (Figure 4e). Conversely, HCAR3 overexpression resulted in increased keratinocyte movement (Figure 4f). Supporting the role for HCAR3 in keratinocyte migration, we observed similar effects of HCAR3 knockdown and overexpression in non-confluent cell cultures (Figure 4g, h, i and j).

Figure 4: HCAR3 expression affects keratinocytes migration.

Figure 4:

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(a) Representative images from scratch assays in N/TERT2G cells transfected with the specified siRNAs for 72hs. The open area is indicated in red color. Scale bar: 100μm. (b) and (c) Quantification of scratch assays in HEK (b) and N/TERT2G (c) cells. The scratch area was quantified at the indicated times and normalized to time zero. n=5 independent experiments performed in triplicates. (d) Quantification of the area under the curve (AUC) for graphs shown on (b) and (c). Data was normalized to siCtrl represented as a black dotted line. n=5 independent experiments performed in triplicates (e) and (f) Quantification of accumulated distance for individual N/TERT2G cells transfected with the specified siRNAs in the scratch assay (e) or iHCAR3 cells treated as indicated (f). Cells were tracked for 24h. n=24-40 cells per condition. (g) and (h) Spider plots (g) and quantification of migration expressed as Euclidian distance (h) for individual N/TERT2G cells transfected with the specified siRNAs. Cells were tracked for 24hs. In spider plots, the path of each cell and its final position are represented by a black line and a red dot, respectively. Blue dots are the center of mass. n=30-78 cells per condition. (i) and (j) Spider plots (i) and Euclidian distance (j) for individual iHCAR3 cells treated as indicated. n=30-78 cells per condition. (k) Representative images of E-cadherin expression in confluent cells (no scratch) or 12hs after scratching. E-cadherin is shown in red, and nuclei in blue. Insets show an amplified image of the area marked with a white square. White lines indicate where membrane localization of E-cadherin was quantified. Scale bars: 10 μm. (l) and (m) Quantification of (k). Profile plots show the fluorescence intensity of E-cadherin along a dotted line as indicated in (k). Peak of the graph indicates the membrane. Data were normalized to the first value along the quantification lines shown as a black dotted line. N=25 cells quantified from 3 independent experiments. In (b), (c), (h), and (j): no asterisk p>0.05; *p<0.05; **p<0.01; ***p< 0.001; ****p< 0.0001; (b), (c) and (d): 2-way ANOVA followed by t-test; (h) and (j): unpaired t-test.

The scratch-wound assay data suggested that HCAR3 may affect cell-to-cell interaction by influencing the detachment of cells from the monolayer (Movie S1 and 2). Further analysis revealed that while HCAR3 knockdown did not alter total levels of E-cadherin, it affected its membrane localization (Figure 4k and l, Figure S4d). In contrast, HCAR3 overexpression led to a reduction of membrane E-cadherin (Figure 4m). Our study highlights a previously unrecognized role for HCAR3 in regulating keratinocyte migration and cell-to-cell attachment.

HCAR3 knockdown limits keratinocyte respiratory capacity

So far, our results indicate that HCAR3 has a broader impact on keratinocyte biology than the other examined GPCRs. HCAR3 is particularly intriguing because it is expressed only in higher primates, and its functions are not well studied (Kapolka and Isom, 2020). To fill this gap, we focused on understanding the mechanisms by which HCAR3 regulates keratinocyte biology. Since HCAR3 is a Gαi-coupled receptor (Offermanns, 2017), we investigated whether HCAR3 effects could be recapitulated by Gαi knockdown. Similar to the effects of siHCAR3, siRNA for Gαi reduced keratinocyte proliferation and migration (Figure S5a, b, c, and d). Gαi has been shown to interact with β-arrestins to regulate migration (Smith et al., 2021). However, the knockdown of arrestins did not affect keratinocyte proliferation or migration (Figure S5e, f, g, and h). Our results indicate that HCAR3 might partially regulate keratinocyte proliferation and migration by activating Gαi but not β-arrestin intracellular signaling pathways.

Given the known role of HCARs in metabolic regulation (Offermanns, 2017), we hypothesized that HCAR3 might also influence keratinocyte proliferation and migration by affecting cellular metabolism. We analyzed gene networks related to metabolism and mitochondria functions in response to HCAR3 knockdown to test this possibility. We found that siHCAR3 resulted in an upregulation of processes related to fatty acid metabolism and a downregulation of amino-acid metabolism and mitochondrial-related processes (Figure 5a).

Figure 5: HCAR3 regulates keratinocyte metabolism.

Figure 5:

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(a) Graphs of GSEA analysis showing the normalized enrichment score for selected GO biological process in siHCAR3 differentially regulated genes. m.p.: metabolic process, mito.:mitochondria (b) Oxygen consumption rates (OCR) normalized to cell number in N/TERT2G cells transfected with a non-targeting siRNA (siCtrl) or siHCAR3 under basal conditions and in response to the indicated drugs (Con: control, Oligom.: oligomycin, R/A: rotenone/antimycin). n= 4-6 replicates. The graph is representative of 2 independent experiments. (c) Diagram showing the OCR values calculated from (b). (d) Percent change in OCR from drug-treated cells with respect to control cells. n= 4-6 replicates. (e) Representative images and quantification of mitochondrial staining with MitoTracker in N/TERT2G cells transfected with the specified siRNAs, or iHCAR3 treated as indicated for 72hs. Insets show an amplified image of the area marked with a white square. Violin plots represent the distribution of the mitochondrial fragmentation count per cell. n=9-24 cells per condition. Scale bar: 20 μm. (f) Representative images and quantification of lipid droplet staining with LipidTOX in N/TERT2G cells transfected with the specified siRNAs. Violin plots represent the distribution of the number of lipid droplets per cell. n=70-300 cells per condition. Scale bar: 20 μm. In (c), (d), (e), and (f): no asterisk p>0.05; *p<0.05; **p<0.01; ****p< 0.0001; C, ANOVA followed by t-test; (d), ANOVA followed by Sidak's multiple comparison test; (e), Mann-Whitney test; (f), unpaired t-test.

To examine the role of HCAR3 in keratinocyte metabolism, we measured oxygen consumption and substrate preference in siHCAR3 cells using a Seahorse Analyzer. We found a drop in the oxygen consumption rate, indicative of a reduction in oxidative phosphorylation (OXPHOS), following HCAR3 knockdown (Figure 5b and c). We next evaluated the effect of siHCAR3 on metabolic substrate by measuring oxygen consumption in the presence of inhibitors of fatty acid oxidation (etomoxir), pyruvate metabolism (UK5099), or glutamine metabolism (BPTES). Knockdown of HCAR3 increased the reliance on fatty acids for OXPHOS, as evidenced by the decrease in respiratory capacity upon etomoxir treatment in siHCAR3 but not in control cells (Figure 5d). These results suggest that HCAR3 may regulate the balance of metabolic substrates utilized by keratinocytes, with knockdown of HCAR3 shifting cells towards increased fatty acid utilization. This shift results in energy defects, reducing respiratory capacity and ATP production.

Consistent with the decreased metabolic state of keratinocytes, siHCAR3 caused mitochondrial fragmentation (Figure 5e), often associated with metabolic stress (Sprenger and Langer, 2019). In contrast, HCAR3 overexpression increased mitochondrial length and connectivity (Figure 5e). Moreover, siHCAR3 led to an increased number of lipid droplets in keratinocytes (Figure 5f), further supporting a potential role for this receptor in lipid metabolism. Overall, our findings demonstrate that HCAR3 plays a critical role in regulating gene networks that balance keratinocyte metabolism and mitochondrial morphology to ensure optimal energy outputs.

Discussion

GPCRs heavily influence epidermal stem cell biology, enabling cells to sense extracellular cues and adapt to environmental changes, maintaining tissue homeostasis (Pedro, Lund and Iglesias-Bartolome, 2020). To better understand the specific GPCRs that regulate epithelial cells, we used primary cultures of human keratinocytes and identified LTB4R, HCAR3, GPR153, and GPR137 as key receptors that regulate epithelial cell proliferation and differentiation. Furthermore, we determined the transcriptional and signaling networks affected by these GPCRs. Our findings provide valuable insights into the complex interplay between GPCRs and epithelial cell fate decisions.

This study shows that LTB4R, GPR153, and GPR137 affect keratinocyte proliferation and differentiation; however, knockout mice for the homologous genes Ltb4r1, Gpr153, and Gpr137 do not have any apparent defects in skin development (Groza et al., 2023, Oyoshi et al., 2012). The lack of an epidermal phenotype in knockout mice shows that these receptors might not be essential for skin homeostasis, although our results suggest that they could have functions during pathological conditions. Indeed, Ltb4r1 knockout mice have reduced epidermal cell proliferation in response to allergic skin inflammation (Oyoshi et al., 2012), indicating that LTB4R can drive keratinocyte proliferation in vivo. Similarly, GPR137 could have a role in hyperproliferative diseases since it can regulate cell growth by interacting with Rag-mTORC1 signaling and induce proliferation in cancer cells (Gan et al., 2019, Mager et al., 2017). The overexpression of LTB4R, GPR137, and HCAR3 in 3D organotypic cultures resulted in abnormal skin development and differentiation, suggesting potential pathological effects due to their upregulation.

Proper coordination of metabolic pathways with nutritional requirements is essential to sustain and restore skin homeostasis after injury (Cibrian, de la Fuente and Sanchez-Madrid, 2020, Eming, Murray and Pearce, 2021). While this highlights the need for epithelial cells to fine-tune their metabolic state, little is known about how keratinocytes integrate environmental signals into intracellular metabolic pathways. GPCRs can mediate the signaling of numerous metabolites (Husted et al., 2017), and the HCAR protein family members regulate cellular metabolism under changing nutrient conditions (Offermanns, 2017). Remarkably, HCAR2 and HCAR3 are some of the most expressed GPCR genes in human keratinocytes (Figure 1B). HCAR2 (also known as GPR109A) is activated by the antidyslipidemic drug nicotinic acid and has anti-inflammatory and potential anti-psoriatic effects in the skin (Hanson et al., 2010, Lukasova et al., 2011). Although the roles of the closely related receptor HCAR3 are not well known due to its exclusive expression in higher primates (Kapolka and Isom, 2020), our study shows that HCAR3 can affect multiple aspects of epithelial cell biology, including proliferation, differentiation, and migration.

In adipocytes, HCAR receptors are part of a negative feedback loop that senses intermediates from fatty acid β-oxidation in the extracellular space and blocks intracellular lipolysis (Offermanns, 2017). This information suggests that HCAR3 could have a similar metabolic role in keratinocytes. Indeed, HCAR3 knockdown reduced OXPHOS, possibly by altering metabolite use and mitochondrial morphology. HCAR3 could affect lipid oxidation and de novo lipogenesis in keratinocytes since HCAR3 knockdown caused lipid droplets to accumulate in cells. HCAR2 can function similarly in other cell types (Lee et al., 2020, Ye et al., 2019), indicating that these receptors regulate multiple aspects of lipid metabolism.

The metabolic switch caused by HCAR3 knockdown led to reduced mitochondrial activity and energy output. Interestingly, proliferating keratinocytes are highly dependent on glucose, and reduction of glucose metabolism can lead to defects in proliferation and migration (Zhang et al., 2018). HCAR3 depletion may disturb the metabolic sensing state of keratinocytes, leading to metabolic imbalances between glycolysis and OXPHOS that result in defects in cell proliferation, differentiation, and migration. Our study highlights the importance of proper coordination between extracellular metabolite signaling and intracellular metabolism in maintaining epithelial cell homeostasis and adapting to stress conditions. Further research into the role of metabolite sensing in epithelial cell fate decisions is needed, offering new possibilities for therapeutic interventions for pathological skin conditions and wound healing.

Finally, while our study identified several GPCRs that regulate keratinocytes, other receptors may have additional functions that can not be identified in cell culture conditions due to the lack of paracrine activation from other cell types or circulating factors. Additional studies are needed to fully understand the complete set of GPCRs involved in regulating keratinocyte biology.

Materials and methods

Detailed materials and methodology are available in the Supplementary Materials and Methods.

Cell culture:

All cells were cultured at 37°C in the presence of 5% CO2. Pooled neonatal primary Human Epidermal Keratinocytes (HEK) were obtained from Life Technologies and used between passages two to five. Under NIH protocols the use of biospecimens from a commercial source does not meet the regulatory criteria for human subject research and therefore does not require IRB review or informed consent, the Common Rule 45 CFR 46 does not apply. N/TERT-2G keratinocyte cell line (Dickson et al., 2000, Smits et al., 2017) was described before (Yuan et al., 2020). Lenti-X 293T cells were obtained from Takara Bio and cultured in DMEM (Sigma-Aldrich) containing 10% fetal bovine serum (Sigma-Aldrich). For GPCR expression analysis, HEK cells were cultured in KGM Keratinocyte Growth Medium BulletKit (Lonza). For all other experiments, HEK and N/TERT-2G keratinocytes were cultured in EpiLife media (Life Technologies) supplemented with Keratinocyte Growth Supplement (S0015, Life Technologies). Where indicated, EpiLife media was supplemented with Defined Growth Supplement (EDGS S0125, Life Technologies. For differentiation culture conditions, cells were plated at 100% confluency in media supplemented with 1.5 mM CaCl2. For siRNA experiments, cells were transfected 24hs after plating and analyzed 72hs after transfection. Cells were transfected with siRNA at 5 pmol/well for 96-well plates and 15 pmol/well for 6-well plates, using Lipofectamine RNAiMAX (Invitrogen). Inducible cell lines were incubated with 0.2 mg/ml of doxycycline to trigger GPCR expression. Human epidermis reconstruction experiments were performed as previously described (Pedro et al., 2020). Cell proliferation was evaluated using 4hs incorporation of EdU and labeling with the Click-IT EdU Imaging Kit (Invitrogen). A Seahorse XFe96 Analyzer and XF Mito Stress Test Kit (Agilent Technologies) were used to measure the oxygen consumption rate (OCR). For scratch assays, cells were plated in 96-well plates and transfected 24 hs later. 72 hrs after transfection, with 100% confluent cells, a scratch was made using a wound maker system (Essen BioScience). Data was collected every 15 min up to 6 hs to 24 hs using a Keyence BZ-X700 microscope equipped with a CO2 and temperature control chamber. The wound area was quantified, and videos created using ImageJ.

DNA constructs:

Codon-optimized sequences for human LTB4R (NP_858043.1), HCAR3 (NP_006009.2), GPR153 (NP_997253.2) and GPR137 (NP_001164351.1) with N-terminal 2xHA tag followed by a GDPPVAT linker were purchased from IDT as gBlocks Gene Fragments and cloned in pInducer20 lentiviral inducible vector. pInducer20 was a gift from Stephen Elledge (Addgene plasmid # 44012). Lentiviruses were produced by transfecting Lenti-X 293T cells with pMD2.VSVG (Addgene plasmid # 12259) and psPAX2 (Addgene plasmid # 12260).

Quantitative PCR and RNA sequencing:

Cells treated as indicated were lysed using the Precellys Lysing Kit (Bertin Instruments). RNA was isolated and processed using RNeasy Plus Mini Kit (Qiagen). 1μg of RNA was used to synthesize cDNA with SensiFAST cDNA Synthesis Kit (Bioline) and used as a template for quantitative polymerase chain reaction with reverse transcription (qRT–PCR) analysis using SensiFAST SYBR Lo-ROX Kit (Bioline). Samples were analyzed using a QuantStudio 3 Real-Time PCR System (ThermoFisher). Values were normalized across samples using RPLP0 expression. For GPCR expression analysis, RNA sequencing was performed in primary human keratinocytes harvested 24h after plating (basal) and at 24h, 48h, and 96h after differentiation in triplicates. For knockdown RNA sequencing experiments, cells were transfected with the corresponding siRNA in triplicate; the following day media was changed, and cells were harvested after 72h. RNA integrity was measured with the TapeStation system (Agilent). Samples with RIN>8 were further processed. mRNA expression profiling was performed in the CCR-Sequencing Facility at the NIH.

Immunoblot and immunofluorescence:

Western blot, immunofluorescence and histology analysis was performed as described previously (Pedro et al., 2020). Quantification of plasma membrane E-cadherin was done in ImageJ using the plot profile tool. HCS LipidTOX Green Neutral Lipid Stain (Thermo Fisher Scientific) was used to analyze lipid droplets. For mitochondria staining, cells were incubated for 30 minutes with 100 nM MitoTracker (Thermo Fisher Scientific) at 37°C. Mitochondrial fragmentation was quantified using ImageJ as described in (Rehman et al., 2012). Images were obtained using either a Leica SP8 confocal microscope with LASX software (Fig. 4k, 5e, 5f, S3a) or a Keyence BZ-X700 widefield fluorescent microscope with automatic stage and focus with BZX software (Fig. 1d, 3e, S3c). For Figs. 3e and S3c, final images are maximum intensity projections from 9 to 15 focal planes performed with the BZX software "Full Focus" feature. The final images were bright contrast adjusted with Adobe Photoshop.

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).

Supplementary Material

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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 011763). This work used the computational resources of the NIH High-Performance Computing Biowulf Cluster. We thank the members of the CCR Sequencing Facility at Frederick National Laboratory for Cancer Research for their help during sample preparation, sequencing, and data processing.

Abbreviations:

GO

Gene ontology

GPCR

G protein-coupled receptor

GPR137

G Protein-Coupled Receptor 137

GPR153

G Protein-Coupled Receptor 153

HCAR2

hydroxycarboxylic acid-receptor 2

HCAR3

hydroxycarboxylic acid-receptor 3

HEK

human epidermal keratinocytes

IPA

Ingenuity pathway analysis

K10

keratin 10

LTB4R

leukotriene B4-receptor 1

NES

normalized enrichment scores

PCA

Principal component analysis

RNAseq

RNA sequencing

siRNA

small interference RNA

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflict of interest statement:

No competing interests to declare.

Data availability statement:

Raw and processed RNAseq data generated for this study are available in the GEO database under accession codes: GSE228631 and GSE228651. Gpr137 and Gpr153 knockout phenotype data was retrieved from the www.mousephenotype.org (Groza et al., 2023). Single cell expression levels for GPCRs in human skin were obtained from v22.proteinatlas.org (Karlsson et al., 2021), data available at: https://www.proteinatlas.org/ENSG00000213903-LTB4R/single+cell+type/skin, https://www.proteinatlas.org/ENSG00000255398-HCAR3/single+cell+type/skin, https://www.proteinatlas.org/ENSG00000173264-GPR137/single+cell+type/skin, https://www.proteinatlas.org/ENSG00000158292-GPR153/single+cell+type/skin.

References

  1. 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]
  2. 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]
  3. Belokhvostova D, Berzanskyte I, Cujba AM, Jowett G, Marshall L, Prueller J, Watt FM. 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]
  4. Blanpain C, Fuchs E. Epidermal homeostasis: a balancing act of stem cells in the skin. Nature reviews Molecular cell biology 2009;10(3):207–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Cibrian D, de la Fuente H, Sanchez-Madrid F. Metabolic Pathways That Control Skin Homeostasis and Inflammation. Trends Mol Med 2020;26(11):975–86. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Eming SA, Murray PJ, Pearce EJ. Metabolic orchestration of the wound healing response. Cell Metab 2021;33(9):1726–43. [DOI] [PubMed] [Google Scholar]
  9. Gan L, Seki A, Shen K, Iyer H, Han K, Hayer A, et al. The lysosomal GPCR-like protein GPR137B regulates Rag and mTORC1 localization and activity. Nature cell biology 2019;21(5):614–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Groza T, Gomez FL, Mashhadi HH, Munoz-Fuentes V, Gunes O, Wilson R, et al. The International Mouse Phenotyping Consortium: comprehensive knockout phenotyping underpinning the study of human disease. Nucleic Acids Res 2023;51(D1):D1038–D45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144(5):646–74. [DOI] [PubMed] [Google Scholar]
  12. Hanson J, Gille A, Zwykiel S, Lukasova M, Clausen BE, Ahmed K, et al. Nicotinic acid- and monomethyl fumarate-induced flushing involves GPR109A expressed by keratinocytes and COX-2-dependent prostanoid formation in mice. J Clin Invest 2010;120(8):2910–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Husted AS, Trauelsen M, Rudenko O, Hjorth SA, Schwartz TW. GPCR-Mediated Signaling of Metabolites. Cell Metab 2017;25(4):777–96. [DOI] [PubMed] [Google Scholar]
  14. Iglesias-Bartolome R, Gutkind JS. Signaling circuitries controlling stem cell fate: to be or not to be. Current opinion in cell biology 2011;23(6):716–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kapolka NJ, Isom DG. HCAR3: an underexplored metabolite sensor. Nat Rev Drug Discov 2020;19(11):745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Karlsson M, Zhang C, Mear L, Zhong W, Digre A, Katona B, et al. A single-cell type transcriptomics map of human tissues. Sci Adv 2021;7(31). [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kobayashi NR, Hawes SM, Crook JM, Pebay A. G-protein coupled receptors in stem cell self-renewal and differentiation. Stem Cell Rev Rep 2010;6(3):351–66. [DOI] [PubMed] [Google Scholar]
  18. Lee AK, Kim DH, Bang E, Choi YJ, Chung HY. beta-Hydroxybutyrate Suppresses Lipid Accumulation in Aged Liver through GPR109A-mediated Signaling. Aging Dis 2020;11(4):777–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell 2013;153(6):1194–217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lukasova M, Malaval C, Gille A, Kero J, Offermanns S. Nicotinic acid inhibits progression of atherosclerosis in mice through its receptor GPR109A expressed by immune cells. J Clin Invest 2011;121(3):1163–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mager LF, Koelzer VH, Stuber R, Thoo L, Keller I, Koeck I, et al. The ESRP1-GPR137 axis contributes to intestinal pathogenesis. Elife 2017;6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Offermanns S. Hydroxy-Carboxylic Acid Receptor Actions in Metabolism. Trends Endocrinol Metab 2017;28(3):227–36. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Pedro MP, Lund K, Iglesias-Bartolome R. The landscape of GPCR signaling in the regulation of epidermal stem cell fate and skin homeostasis. Stem Cells 2020;38(12):1520–31. [DOI] [PubMed] [Google Scholar]
  25. Pedro MP, Salinas Parra N, Gutkind JS, Iglesias-Bartolome R. Activation of G-Protein Coupled Receptor-Galphai Signaling Increases Keratinocyte Proliferation and Reduces Differentiation, Leading to Epidermal Hyperplasia. The Journal of investigative dermatology 2020;140(6):1195–203 e3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nature reviews Molecular cell biology 2002;3(9):639–50. [DOI] [PubMed] [Google Scholar]
  28. Pullar CE, Le Provost GS, O'Leary AP, Evans SE, Baier BS, Isseroff RR. beta2AR antagonists and beta2AR gene deletion both promote skin wound repair processes. The Journal of investigative dermatology 2012;132(8):2076–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rehman J, Zhang HJ, Toth PT, Zhang Y, Marsboom G, Hong Z, et al. Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2012;26(5):2175–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Sivamani RK, Shi B, Griffiths E, Vu SM, Lev-Tov HA, Dahle S, et al. Acute wounding alters the beta2-adrenergic signaling and catecholamine synthetic pathways in keratinocytes. The Journal of investigative dermatology 2014;134(8):2258–66. [DOI] [PubMed] [Google Scholar]
  33. Smith JS, Pack TF, Inoue A, Lee C, Zheng K, Choi I, et al. Noncanonical scaffolding of G(alphai) and beta-arrestin by G protein-coupled receptors. Science 2021;371(6534). [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Sprenger HG, Langer T. The Good and the Bad of Mitochondrial Breakups. Trends Cell Biol 2019;29(11):888–900. [DOI] [PubMed] [Google Scholar]
  36. Watt FM. Mammalian skin cell biology: at the interface between laboratory and clinic. Science 2014;346(6212):937–40. [DOI] [PubMed] [Google Scholar]
  37. Ye L, Cao Z, Lai X, Wang W, Guo Z, Yan L, et al. Niacin fine-tunes energy homeostasis through canonical GPR109A signaling. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2019;33(4):4765–79. [DOI] [PubMed] [Google Scholar]
  38. Yuan Y, Park J, Feng A, Awasthi P, Wang Z, Chen Q, Iglesias-Bartolome R. YAP1/TAZ-TEAD transcriptional networks maintain skin homeostasis by regulating cell proliferation and limiting KLF4 activity. Nat Commun 2020;11(1):1472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang Z, Zi Z, Lee EE, Zhao J, Contreras DC, South AP, et al. Differential glucose requirement in skin homeostasis and injury identifies a therapeutic target for psoriasis. Nature medicine 2018;24(5):617–27. [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
NIHMS1952988-supplement-1.tif (8.4MB, tif)
2
NIHMS1952988-supplement-2.tif (18.5MB, tif)
3
NIHMS1952988-supplement-3.tif (15.9MB, tif)
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NIHMS1952988-supplement-4.tif (5.1MB, tif)
5
NIHMS1952988-supplement-5.tif (3.6MB, tif)
6
Download video file (30.1MB, mp4)
7
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NIHMS1952988-supplement-8.docx (45.6KB, docx)
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NIHMS1952988-supplement-9.xlsx (30.8KB, xlsx)
10
NIHMS1952988-supplement-10.xlsx (3.7MB, xlsx)
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NIHMS1952988-supplement-11.xlsx (2.4MB, xlsx)

Data Availability Statement

Raw and processed RNAseq data generated for this study are available in the GEO database under accession codes: GSE228631 and GSE228651. Gpr137 and Gpr153 knockout phenotype data was retrieved from the www.mousephenotype.org (Groza et al., 2023). Single cell expression levels for GPCRs in human skin were obtained from v22.proteinatlas.org (Karlsson et al., 2021), data available at: https://www.proteinatlas.org/ENSG00000213903-LTB4R/single+cell+type/skin, https://www.proteinatlas.org/ENSG00000255398-HCAR3/single+cell+type/skin, https://www.proteinatlas.org/ENSG00000173264-GPR137/single+cell+type/skin, https://www.proteinatlas.org/ENSG00000158292-GPR153/single+cell+type/skin.

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