OR2AT4, an Ectopic Olfactory Receptor, Suppresses Oxidative Stress-Induced Senescence in Human Keratinocytes

Olfactory receptors (ORs) are the largest protein superfamily in mammals. Certain ORs are ectopically expressed in extranasal tissues and regulate cell type-specific signal transduction pathways. OR2AT4 is ectopically expressed in skin cells and promotes wound healing and hair growth. As the capacities of wound healing and hair growth decline with aging, we investigated the role of OR2AT4 in the aging and senescence of human keratinocytes. OR2AT4 was functionally expressed in human keratinocytes (HaCaT) and exhibited co-expression with G-protein-coupled receptor signaling components, Golfα and adenylate cyclase 3. The OR2AT4 ligand sandalore modulates the intracellular calcium, inositol phosphate, and cyclic adenosine monophosphate (cAMP) levels. The increased calcium level induced by sandalore was attenuated in cells with OR2AT4 knockdown. OR2AT4 activation by sandalore inhibited the senescent cell phenotypes and restored cell proliferation and Ki-67 expression. Sandalore also inhibited the expression of senescence-associated β-galactosidase and increased p21 expression in senescent HaCaT cells in response to hydrogen peroxide. Additionally, sandalore activated the CaMKKβ/AMPK/mTORC1/autophagy signaling axis and promoted autophagy. OR2AT4 knockdown attenuated the increased in the intracellular calcium level, cell proliferation, and AMPK phosphorylation induced by sandalore. These findings demonstrate that the effects of sandalore are mediated by OR2AT4 activation. Our findings suggest that OR2AT4 may be a novel therapeutic target for anti-aging and anti-senescence in human keratinocytes.


Introduction
Aging is progressive physiological alterations in an organism that lead to senescence, or a decline of biological functions and of the organism's ability in response to metabolic stress. Aging reduces health span and physiological functions primarily due to the accumulation of cellular damage, thus increasing the risk for developing chronic diseases [1].
According to the progression of aging, human skin is constantly exposed to internal and external stimuli that have an impact on its functionality, manifesting as wrinkling, dry skin, a weakened skin barrier, loss of skin integrity, epidermal thinning, and skin cancer [2][3][4]. Skin aging is regulated by multiple proteins, and it is possible that ectopic olfactory receptors (ORs) may play critical roles since the skin is constantly exposed to external odorants. ORs belong to the largest protein superfamily of guanine nucleotidebinding protein (G protein)-coupled receptors (GPCRs) and are primarily expressed in the cilia of olfactory sensory neurons. These ectopic ORs have been shown to regulate cellular and tissue-specific functions [5,6] thus aroma compounds that are ligands for ectopic ORs may have biological activities in skin.

Cell Culture
The HaCaT human skin keratinocyte line was purchased from the American Type Culture Collection (Manassas, VA, USA). HaCaT Cells were cultured in Dulbecco's Modified Eagle Medium (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Hyclone) in an incubator at 5% CO 2 and 37 • C.

Real-Time Polymerase Chain Reaction (RT-PCR) and Quantitative RT-PCR (qRT-PCR) Analyses
RT-PCR and qRT-PCR analyses were performed, as described previously [5]. Briefly, the total mRNA was extracted using RNAiso Plus (TaKaRa Bio Inc., Shiga, Japan), and the cDNA samples were obtained using ReverTra Ace qPCR RT Master Mix with a gDNA Eraser (Toyobo, Osaka, Japan), according to the manufacturer's instructions. The RT-PCR amplification process was performed using Dreamtaq Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and S1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The RT-PCR products were confirmed by agarose gel electrophoresis at a current of 5 V/cm. The images were captured using the ChemiDoc Touch System (Bio-Rad Laboratories). qRT-PCR was conducted using Thunderbird SYBR ® qPCR Mix reagent (TaKaRa) and was analyzed using the iQ5 Cycler System (Bio-Rad Laboratories). The mRNA expression was validated by the RT-PCR and qRT-PCR analysis of the ribosomal protein L32. The 2 −∆∆Ct method was used to analyze the relative changes in gene expression. The primer sequences used for the experiment are shown in Table 1. GNAS, G protein G(s) subunit α; GNAQ, G protein G(q) subunit α; GNAI1, G protein G(i) subunit α-1; GNA12, G protein subunit α-12; GNA13, G protein subunit α-13; ADCY3, adenylate cyclase 3; L32, ribosomal protein L32.

Cre-Luciferase Assay
For the Cre-luciferase assay, the Hana3A cells were grown in MEM containing 10% FBS with 1% PEST [22]. Hana3A cells were seeded 2 × 10 4 cells per well in 96-well plates. The Hana3A cells were transfected with OR2AT4 (OriGene, Rockville, MD, USA) and an empty vector as the control using the DharmaFECT transfection reagent (Horizon Discovery, Cambridge, UK), and the transfection process was conducted by following the manufacturer's guidelines. Subsequently, the control group was treated with DMSO, the positive control group was treated with 100 µM forskolin (FSK; Cayman), and the experimental group was treated with 50 and 100 µM sandalore (Givaudan Schweiz AG, Dübendorf, Switzerland) for 24 h. Luciferase activities were detected by the dual-luciferase reporter assay system (E1960, Promega, Madison, WI, USA) and VICTOR ™ X Multilabel Plate Reader (PerkinElmer, Inc., Waltham, MA, USA).

cAMP Assay
For the intracellular cAMP assay, HaCaT cells were seeded in a 96-well plate at a density of 4 × 10 4 /well. After 24 h of incubation, the control group was treated with DMSO, the positive control group was treated with 100 µM forskolin, and the experimental group was treated with sandalore (50 or 100 µM) for 24 h. A screen quest fluorometric ELISA cAMP assay kit (36373; AAT Bioquest, Sunnyvale, CA, USA) and VICTOR ™ X Multilabel Plate Reader (PerkinElmer, Inc.) were used to determine the cAMP concentration changes, according to the manufacturers' instructions.

Inositol Phosphate Assay
For the intracellular inositol phosphate assay, HaCaT cells were seeded in a 384-well OptiPlate (PerkinElmer, Inc.) at a density of 4 × 10 4 /well. After overnight incubation, the control group was treated with DMSO and the experimental group was treated with 50 and 100 µM sandalore for 24 h. The inositol phosphate concentration changes were detected using the IP-One Tb kit ® (62IPAPEC; CisBio Bioassays, Codolet, France) and EnVision Xcite Multilabel plate reader (PerkinElmer, Inc.) equipped with homogenous time-resolved fluorescence filters. The homogenous time-resolved fluorescence ratio was calculated by dividing the raw fluorescence values measured at 665 nm by the raw signals measured at 620 nm.

Calcium Assay
For the intracellular calcium assay, the HaCaT cells were seeded in a 96-well plate at a density of 4 × 10 4 /well. After overnight incubation, the control group was treated with DMSO, the positive control group was treated with A23187 (10 µM; Cayman chemical), and the experimental group was treated with sandalore (100 µM, 300 µM, 1 mM, and 3 mM) for 24 h. Calcium concentration changes were detected using the fluo-4 NW calcium assay kit (F36206; Molecular Probes, Eugene, OR, USA) and VICTOR ™ X Multilabel Plate Reader (PerkinElmer, Inc.).

Small Interfering RNA (siRNA) Transfection
Human OR2AT4 siRNAs were transfected using a commercial siRNA reagent system (sc-45064; Santa Cruz Biotechnology), following the manufacturer's instructions. Briefly,

Induction of Senescence in HaCaT Cells
For the induction of premature senescence, For the induction of premature senescence, HaCaT cells were treated with 200 µM H 2 O 2 (Sigma-Aldrich, St. Louis, MO, USA) for 3 days. The cells were pretreated with 50 or 100 µM sandalore for 1 h before the addition of H 2 O 2 . The positive control was treated with 25 µM quercetin (Sigma-Aldrich).

Senescence-Associated β-Galactosidase (SA-β-gal) Staining
SA-β-gal staining was performed according to a previously published protocol [23]. At pH 6.0, positive staining of β-galactosidase has been reported to remarkably increase in senescent cells. Briefly, the cells were washed twice with PBS and fixed with 0.2% glutaraldehyde for 15 min at room temperature. The fixed cells were washed with PBS and incubated with a freshly prepared SA-β-gal staining solution (1 mg/mL X-gal, 5 mM K 3 FeCN 6 , 5 mM K 4 FeCN 6 , and 2 mM MgCl 2 ) overnight at 37°C without CO 2 . The positive blue-stained cells were counted under a microscope at 100× magnification and expressed as a percentage using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cell Counting and Proliferation Assay
The HaCaT cells were treated with DMSO as a control, quercetin (25 µM), and sandalore (50 and 100 µM) for 1 h, and then co-treated with 200 µM H 2 O 2 for 72 h. The cells were detached by trypsinization for counting. The total viable cells were counted using the automated cell counter Adam-MC with the Adam MC AccuChip 4× kit (NanoEn-Tek Inc., Seoul, Korea). Cell proliferation was measured using the CyQUANT ® NF Cell Proliferation Assay Kit (Invitrogen), according to the manufacturer's instructions. Cell proliferation was measured based on fluorescence using the VICTOR TM X Multilabel Plate Reader (PerkinElmer) at an excitation wavelength of 485 nm and emission wavelength of 535 nm.

Monodansylcadaverine (MDC) Staining
For MDC staining, the cells were treated with 50 µM MDC (Sigma-Aldrich) in the medium and were incubated at 37 • C for 20 min. The MDC-stained autophagic vacuoles were examined by fluorescence microscopy. Images were obtained using the LSM510 META confocal microscope and were analyzed using LSM700 software (version 3.2; Carl Zeiss, Jena, Germany). The MDC-stained cells were counted using ImageJ software (National Institutes of Health).

Statistical Analysis
Data are expressed as means ± standard error of the mean (SEM) based on at least triplicate experiments. Student's t-test was used to compare the groups. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). A p-value < 0.05 was considered statistically significant.

OR2AT4 and OR Signaling Components Are Expressed in Human Keratinocytes
OR2AT4 is ectopically expressed in human primary keratinocytes, and sandalore is a ligand of OR2AT4 [6]. We confirmed the expression of OR2AT4 and its signaling components in HaCaT human keratinocytes ( Figure 1A,B). G proteins play crucial roles in signal transmission via membrane-bound receptors along cell membranes [24,25]. The RT-PCR and RT-qPCR analyses showed that OR2AT4, GNAL, and ADCY3 are co-expressed in HaCaT cells ( Figure 1A,B). GNAL is a G protein isoform that interacts with ORs, and ADCY3 produces cAMP as an effector of GNAL [24]. The RT-PCR results demonstrated that GNAQ, GNAS, GNAI1, GNAI1, GNA12, and GNA13 were also expressed in HaCaT cells.
Immunocytochemistry demonstrated that OR2AT4 is colocalized with Na + /K + -ATPase, a cell surface marker ( Figure 1C). Immunoblotting revealed that OR2AT4, GNAL, and ADCY3 proteins are expressed in the membrane fraction of HaCaT cells ( Figure 1D). These results suggest that OR2AT4 is expressed on the surface membrane of OR2AT4 together with OR signaling components.
PCR and RT-qPCR analyses showed that OR2AT4, GNAL, and ADCY3 are co-expressed in HaCaT cells ( Figure 1A,B). GNAL is a G protein isoform that interacts with ORs, and ADCY3 produces cAMP as an effector of GNAL [24]. The RT-PCR results demonstrated that GNAQ, GNAS, GNAI1, GNAI1, GNA12, and GNA13 were also expressed in HaCaT cells. Immunocytochemistry demonstrated that OR2AT4 is colocalized with Na + /K + -ATPase, a cell surface marker ( Figure 1C). Immunoblotting revealed that OR2AT4, GNAL, and ADCY3 proteins are expressed in the membrane fraction of HaCaT cells ( Figure 1D). These results suggest that OR2AT4 is expressed on the surface membrane of OR2AT4 together with OR signaling components.

Activation of OR2AT4 by Sandalore in Human Keratinocytes
Next, we investigated the second messengers induced by sandalore in HaCaT cells. Sandalore consists of a 2,2,3-trimethylcyclopent-3-enyl structure with a polar OH group (Figure 2A) and is a ligand for OR2AT4 [6]. In the CRE-luciferase assay using OR2AT4transfected Hana3A cells [22], sandalore significantly activated OR2AT4 ( Figure 2B). In the second messenger analysis, sandalore treatment induced all three second messengers, i.e., cAMP ( Figure 2C), inositol phosphate ( Figure 2D), and calcium ( Figure 2E), in HaCaT cells. To determine whether the sandalore effect depends on OR2AT4, we knocked down
To determine whether the sandalore effect depends on OR2AT4, we knocked down the OR2AT4 gene. The mRNA expression of OR2AT4 was reduced by approximately 70% in the cells transfected with OR2AT4 siRNA ( Figure 2F). The increase in the calcium level in HaCaT cells by sandalore was attenuated by OR2AT4 knockdown ( Figure 2G). Our results indicate that OR2AT4 activation by sandalore stimulates complex secondary messenger responses, and that the increase in the intracellular calcium level depends on OR2AT4.
Antioxidants 2022, 11, x FOR PEER REVIEW 7 of 17 the OR2AT4 gene. The mRNA expression of OR2AT4 was reduced by approximately 70% in the cells transfected with OR2AT4 siRNA ( Figure 2F). The increase in the calcium level in HaCaT cells by sandalore was attenuated by OR2AT4 knockdown ( Figure 2G). Our results indicate that OR2AT4 activation by sandalore stimulates complex secondary messenger responses, and that the increase in the intracellular calcium level depends on OR2AT4.

OR2AT4 Activation by Sandalore Recovers the Proliferation Capacity of H 2 O 2 -Induced Senescent HaCaT Cells
Aging is a complex biological process characterized by cellular senescence. To understand the role of OR2AT4 in the aging of human keratinocytes, we investigated whether  Figure 3A). After the induction of senescence by H 2 O 2 , the SA-β-gal expression was evident in HaCaT cells ( Figure 3B). H 2 O 2 -treated HaCaT cells were enlarged, flattened, and irregularly shaped, which are characteristics of senescent cells, suggesting that HaCaT cells underwent senescence ( Figure 3B; left panel). The quantification of SA-β-gal expression showed that the proportion of SA-β-gal positive cells was significantly higher in senescent cells compared with cells that were not treated with H 2 O 2 , whereas sandalore significantly decreased the proportion of SA-β-gal-positive cells ( Figure 3B, right panel). These results demonstrate that sandalore effectively reduced senescence in HaCaT keratinocytes. Next, we examined the effect of sandalore on cell proliferation. The viable cell counting and CyQUANT cell proliferation assays showed that sandalore significantly increased proliferation in the H 2 O 2 -induced senescent cells ( Figure 3C,D). The effects of sandalore were attenuated in senescent cells with OR2AT4 knockdown ( Figure 3E). These results indicate that sandalore recovers the proliferation capacity of senescent cells in an OR2AT4-dependent manner. Next, we assessed the expression of p21 and Ki-67. p21 is a pleiotropic inhibitor of Next, we assessed the expression of p21 and Ki-67. p21 is a pleiotropic inhibitor of the cyclin/cyclin-dependent kinase complexes that mediate cell cycle progression. The induction of p21 causes cell cycle arrest in senescent cells [28]. Ki-67 is a nuclear protein that is highly expressed in asynchronously cycling cells and is absent in non-dividing cells [29]. It is a common marker of proliferating cells [30]. The H 2 O 2 -induced senescent cells expressed a higher expression of p21 mRNA compared with non-senescent cells ( Figure 4A). Quercetin, a positive control, reduced the p21 mRNA expression in nonsenescent and senescent cells. Sandalore did not affect the p21 expression in non-senescent cells, but significantly suppressed the p21 expression in senescent cells ( Figure 4A). The immunoblotting analysis showed similar trends for p21 protein and mRNA expression ( Figure 4B).
Antioxidants 2022, 11, x FOR PEER REVIEW 10 of 17 the proliferation of senescent cells but not non-senescent cells, which is in line with the p21 expression results ( Figure 4A,B). The immunocytochemical analysis showed that the Ki-67 expression was significantly increased in senescent HaCaT cells. Ki-67-positive cells were significantly increased in the cells treated with sandalore compared with the control senescent cells (p < 0.01; Figure 4D). Importantly, the sandalore induction of the Ki-67 mRNA expression in HaCaT cells was attenuated by OR2AT4 knockdown ( Figure 4E). Taken together, our results demonstrate that sandalore treatment recovers cell proliferation by regulating p21 and Ki-67 in HaCaT cells via OR2AT4-dependent mechanisms.  The Ki-67 mRNA expression was lower in senescent cells than non-senescent cells ( Figure 4C). Sandalore significantly increased the Ki-67 gene expression in senescent cells. Additionally, quercetin increased the Ki-67 mRNA expression in non-senescent and senescent cells. Sandalore selectively increased the Ki-67 expression in senescent cells, but not non-senescent cells ( Figure 4C). These findings suggest that sandalore may recover the proliferation of senescent cells but not non-senescent cells, which is in line with the p21 expression results ( Figure 4A,B). The immunocytochemical analysis showed that the Ki-67 expression was significantly increased in senescent HaCaT cells. Ki-67-positive cells were significantly increased in the cells treated with sandalore compared with the control senescent cells (p < 0.01; Figure 4D). Importantly, the sandalore induction of the Ki-67 mRNA expression in HaCaT cells was attenuated by OR2AT4 knockdown ( Figure 4E). Taken together, our results demonstrate that sandalore treatment recovers cell proliferation by regulating p21 and Ki-67 in HaCaT cells via OR2AT4-dependent mechanisms.

OR2AT4 Activation by Sandalore Stimulates the CaMKKβ/AMPK/mTORC1 Signaling Pathway to Suppress Senescence in HaCaT Cells
To explore the mechanisms underlying the suppression of senescence by OR2AT4, we investigated the cellular signaling pathway regulated by sandalore-induced OR2AT4 activation in HaCaT cells. Sandalore increased the intracellular calcium concentration in an OR2AT4-dependent manner. We performed immunoblotting to evaluate CaMKKβ phosphorylation, which is activated by intracellular calcium. Cytosolic calcium stimulates the CaMKKβ signaling cascade, leading to several cellular responses [31,32]. Furthermore, activated CaMKKβ phosphorylates AMPK [32]. Sandalore increased the phosphorylation of CaMKKβ and AMPK in both non-senescent and senescent cells ( Figure 5A,B). In OR2AT4-knockdown cells, the effect of sandalore on AMPK phosphorylation was attenuated ( Figure 5C). Our results demonstrated that sandalore increased the phosphorylation of CaMKKβ and its downstream target AMPK in an OR2AT4-dependent manner ( Figure 5A,B).
AMPK activation stimulates autophagy [33,34], which can be assessed in vitro by MDC staining [35]. Thus, we evaluated the induction of autophagy by sandalore using MDC, an auto-fluorescent dye, in HaCaT cells. We found that sandalore increased autophagy in senescent HaCaT cells ( Figure 5C). During autolysosome formation, LC3II is processed from the cytosolic form (LC3I) to the membrane-bound form (LC3II), thus the LC3II/LC3I ratio is a key molecular marker of autophagy [36]. In the immunoblot analysis, the induction of senescence by H 2 O 2 reduced the LC3II/LC3I ratio compared with that in non-senescent cells, suggesting the inhibition of autophagy in senescent cells. Rapamycin, a known autophagy inducer, increased the LC3II/LC3I ratio in non-senescent and senescent cells. OR2AT4 activation by sandalore (100 µM) also increased the LC3II/LC3I ratio in nonsenescent and senescent cells ( Figure 5D). These findings suggest that sandalore activates cellular autophagy in non-senescent and senescent cells. . Bar graphs represent the ratio of the LC3II to LC3I within each sample (right panel). (E) Expression of the total and phosphorylated mTORC1 and 70S6K by immunoblot analysis. (F) Expression of the total and phosphorylated AMPK in the OR2AT4knockdown HaCaT cells. The relative protein expression of p-CAMKKβ/CAMMKβ, p-AMPK/AMPK, p-mTORC1/TORC1, and p-p70S6K/p70S6K were analyzed using ImageLab software. Data are means ± SEM. * p < 0.05, ** p <0.01, and *** p < 0.001. Student's t-test was performed for comparisons between groups.

Discussion
The skin is the largest organ and forms a mechanical barrier that senses numerous external chemicals via the cell surface sensory receptors, such as ectopic ORs [39,40]. (F) Expression of the total and phosphorylated AMPK in the OR2AT4-knockdown HaCaT cells. The relative protein expression of p-CAMKKβ/CAMMKβ, p-AMPK/AMPK, p-mTORC1/TORC1, and p-p70S6K/p70S6K were analyzed using ImageLab software. Data are means ± SEM. * p < 0.05, ** p <0.01, and *** p < 0.001. Student's t-test was performed for comparisons between groups.
One of the major mechanisms of AMPK-dependent autophagy activation is the inhibition of mTORC1 phosphorylation. In the immunoblot analysis, the senescent cells showed increased phosphorylation of mTORC1 and its target protein p70S6K compared with the non-senescent cells ( Figure 5D). Rapamycin and sandalore decreased the phosphorylation of mTORC1 and p70S6K ( Figure 5E). These results demonstrate that OR2AT4 activation by sandalore stimulates the CaMKKβ/AMPK/mTORC1/autophagy signaling axis. It has been reported that the activation of the CaMKKβ/AMPK/mTORC1 signaling axis can suppress aging and senescence in multiple cells and tissues [37,38].

Discussion
The skin is the largest organ and forms a mechanical barrier that senses numerous external chemicals via the cell surface sensory receptors, such as ectopic ORs [39,40]. Ectopic ORs play significant roles in several tissues, including keratinocytes and other skin cells [6,16,41]. For example, the function of OR2AT4 has been evaluated in skin cells including keratinocytes [6,16,42]. OR2AT4 activation increased human keratinocyte proliferation, migration, and regeneration in vitro, as well as wound re-epithelialization and hair growth ex vivo [6]. OR2AT4 expression in keratinocytes promotes wound healing and hair growth, of which the activity declines with aging. Therefore, we hypothesized that OR2AT4 may suppress aging and senescence in keratinocytes. In the present study, we investigated whether OR2AT4 could regulate cellular aging and senescence in human keratinocytes. To understand the functions of OR2AT4 in aging and the underlying mechanisms, we added sandalore, a ligand of OR2AT4, to H 2 O 2 -induced premature senescent HaCaT human keratinocytes. First, we confirmed the expression of OR2AT4 and OR signaling components, including GNAL and ADCY3, in HaCaT cells. Calcium plays important roles in regulating the sensitivity of olfactory transduction as part of odor adaptation, because odorant adaptation involves modulation of the cAMP-gated channel by calcium feedback [43]. OR2AT4 activation by sandalore increased the intracellular calcium, cAMP, and inositol phosphate levels, whereas OR2AT4 knockdown attenuated the increased calcium level, suggesting that sandalore increased the calcium level via OR2AT4 activation.
Human skin senescence is a progressive process that involves intrinsic or extrinsic aging [39]; increased susceptibility to wrinkles; and decreased self-healing properties, dermal elasticity, and epidermal barrier maintenance [2][3][4]. Skin senescence, which is mediated by time and environmental factors, proceeds via a common molecular pathway that involves ROS formation [3,4,44,45]. In human cells, H 2 O 2 is naturally produced during many physiologic and pathological processes. It is commonly used as a model pro-oxidant to evaluate oxidative stress and premature senescence [21,27]. Stress-induced premature senescence in response to H 2 O 2 exposure involves different pathways than those of replicative senescence [21,26,27]. In our study, OR2AT4 activation ameliorated SA β-galactosidase activity and increased cell proliferation and Ki-67 expression, a marker of cell proliferation. Quercetin treatment was also associated with significantly increased cell proliferation in non-senescent and senescent cells; however, sandalore increased cell proliferation in senescent cells, but not in non-senescent cells. These findings suggest that sandalore and OR2AT4 may recover cell proliferation activity particularly in senescent cells, without affecting normal cells, and may be beneficial for clinical applications. Our results demonstrate that OR2AT4 activation by sandalore suppresses the expression of p21, a senescence-associated secretory phenotype factor and cell cycle arrest factor, in senescent cells. p21 is involved in cell cycle arrest in the G1 phase of senescent cells [46]. Additionally, p21 activates P53-induced cell cycle arrest or apoptosis [47]. Cellular senescence and cell cycle arrest involve essential checkpoints of cellular homeostasis, such as the maintenance of DNA replication, repair, and division [48,49]. Increased cell proliferation by sandalore in senescent cells was attenuated by OR2AT4 knockdown. Therefore, increased cell proliferation and Ki67 expression, as well as decreased SA-β gal and p21 expression, in senescent cells demonstrated that sandalore may recover the cell proliferation capacity of senescent cells via an OR2AT4-dependent mechanism.
Calcium, a key second messenger, activates diverse signal transduction pathways, including the calmodulin kinase pathway. Calmodulin kinase, a Ser/Thr kinase, phosphorylates the Thr residue in the activation loop of CaMKKβ, which further activates AMPK by phosphorylating the 172 Thr of the alpha subunit. AMPK is a critical regulator of cellular energy metabolism, proliferation, and autophagy. Intracellular calcium signaling activates AMPK and autophagy [34]. In the present study, sandalore increased the phosphorylation of CaMKKβ and AMPK, which was suppressed by senescence. The increased AMPK phosphorylation by sandalore was inhibited in cells with OR2AT4 knockdown. These findings suggest that OR2AT4 activation by sandalore enhanced the calcium/CaMKKβ/AMPK signaling axis.
The mTORC1 signaling pathway is one of the major pathways that increases autophagy. AMPK inhibits mTORC1 directly via the phosphorylation of raptor, a component of the mTORC1 complex, and via the phosphorylation of TSC1/2, an inhibitor of mTORC1 [50]. Inhibition of mTORC1 reduces protein synthesis and promotes autophagy [33].
Our results suggest that sandalore decreased the phosphorylation of mTORC1 and P70S6K, a downstream target of mTORC1, in the H 2 O 2 -induced senescence of human keratinocytes. Autophagosome formation was markedly increased in H 2 O 2 -induced senescent keratinocytes via OR2AT4 activation by sandalore treatment. The autophagy content was measured by both MDC staining and immunoblotting of the LC3II/LC3I ratio, respectively. Both results consistently demonstrated that autophagy was increased through the activation of OR2AT4 by sandalore. Autophagy is generally reduced in cells under pathological conditions, such as aging [51]. Therefore, OR2AT4 activation by sandalore increased autophagosome formation, at least partly, via the calcium/CaMKKβ/AMPK/mTORC1 signaling axis. mTORC1 signaling increases the senescence-associated secretory phenotype gene expression, and the expression of p21 is increased by the mTORC1/P70S6K pathway [52,53]. Our results showed that the p21 expression is reduced by sandalore in senescent cells, suggesting that sandalore suppressed the p21 expression by inhibiting the mTORC1/P70S6K pathway via AMPK activation in senescent cells. On the other hand, P70S6K phosphorylation was increased by sandalore in non-senescent cells, while the upstream kinase mTORC1 was suppressed. The reasons for the unexpected results are unclear. It is possible that sandalore may have marginal effects on normal non-senescent cells or have additional direct effects on P79S6K in an mTORC1-independent manner.

Conclusions
This is the first study to demonstrate that OR2AT4 activation by sandalore inhibits H 2 O 2 -induced senescence of human keratinocytes. OR2AT4 activation by sandalore induced the secondary messengers, including calcium, phosphorylated AMPK, and cell proliferation in senescent cells, of which the effects were negated in the gene knockdown experiments. The suppression of senescence by OR2AT4 activation was confirmed by evaluating the expression of several senescence markers, including SA-β-gal staining, cell proliferation rate, Ki67, and p21, in H 2 O 2 -induced senescent keratinocytes. OR2AT4 activation by sandalore restored cell proliferation via activation of the CaMKKβ/AMPK/mTORC1/ autophagy signaling axis ( Figure 6). Our results suggest that OR2AT4 may be a possible therapeutic target for suppressing the aging and senescence of keratinocytes.  Proposed role of OR2AT4 in the suppression of senescence in human keratinocytes. Sandalore binds and activates OR2AT4 to increase the levels of second messengers, mainly intracellular calcium. The increased intracellular calcium level activates the CAMKKβ/AMPK/mTORC1/autophagy signaling axis. OR2AT4 inhibits p21, a marker of cellular senescence, by activating AMPK phosphorylation. Overall, OR2AT4 activation by sandalore may improve cellular senescence in human keratinocytes.

Data Availability Statement:
The data presented in this study are available in article.

Conflicts of Interest:
The authors declare no conflict of interest.