MicroRNA-7a2 Contributes to Estrogen Synthesis and Is Modulated by FSH via the JNK Signaling Pathway in Ovarian Granulosa Cells

MicroRNA-7a2 (miR-7a2) plays fundamental roles in the female reproductive axis, and estrogen is indispensable for maintaining ovary function. However, the interaction between miR-7a2 and ovarian function is unclear. The present study aimed to determine whether and how miR-7a2 functions in estrogen synthesis. Firstly, the results verified that miR-7a was highly expressed in ovarian granulosa cells. The knockout (KO) of miR-7a2 caused infertility and abnormal ovarian function in mice. Concomitantly, the Cyp19a1 expression and estrogen synthesis were significantly inhibited, which was validated in primary granulosa cells. The mice transplanted with miR-7a2 KO ovaries showed similar results; however, estrogen supplementation reversed infertility. In the in vitro experiment, follicle-stimulating hormone (FSH) significantly improved the expression of miR-7a and Cyp19a1 and the synthesis of estrogen. However, the miR-7a2 KO markedly reversed the function of FSH. Also, FSH upregulated miR-7a by activating the (c-Jun N-terminal kinase) JNK signaling pathway. In addition, Golgi apparatus protein 1 (Glg1) was shown to be the target gene of miR-7a2. These findings indicated that miR-7a2 is essential for ovarian functions with respect to estrogen synthesis through the targeted inhibition of the expression of Glg1 and then promoting Cyp19a1 expression; the physiological process was positively regulated by FSH via the JNK signaling pathway in granulosa cells.     

The Hedgehog-GLI Pathway Regulates MEK5-ERK5 Expression and Activation in Melanoma Cells

Malignant melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. We recently showed that the extracellular signal-regulated kinase 5 (ERK5), encoded by the MAPK7 gene, plays a pivotal role in melanoma by regulating cell functions necessary for tumour development, such as proliferation. Hedgehog-GLI signalling is constitutively active in melanoma and is required for proliferation. However, no data are available in literature about a possible interplay between Hedgehog-GLI and ERK5 pathways. Here, we show that hyperactivation of the Hedgehog-GLI pathway by genetic inhibition of the negative regulator Patched 1 increases the amount of ERK5 mRNA and protein. Chromatin immunoprecipitation showed that GLI1, the major downstream effector of Hedgehog-GLI signalling, binds to a functional non-canonical GLI consensus sequence at the MAPK7 promoter. Furthermore, we found that ERK5 is required for Hedgehog-GLI-dependent melanoma cell proliferation, and that the combination of GLI and ERK5 inhibitors is more effective than single treatments in reducing cell viability and colony formation ability in melanoma cells. Together, these findings led to the identification of a novel Hedgehog-GLI-ERK5 axis that regulates melanoma cell growth, and shed light on new functions of ERK5, paving the way for new therapeutic options in melanoma and other neoplasms with active Hedgehog-GLI and ERK5 pathways.     

Intermittent Hypoxia on the Attenuation of Induced Nasal Allergy and Allergic Asthma by MAPK Signaling Pathway Downregulation in a Mice Animal Model

Intermittent hypoxia (IH) has been an issue of considerable research in recent years and triggers a bewildering array of both detrimental and beneficial effects in several physiological systems. However, the mechanisms leading to the effect are not yet clear. Consequently, we investigated the effects of IH on allergen-induced allergic asthma via the mitogen-activated protein kinase (MAPK) signaling pathway. Forty BALB/c mice were dived into four groups. We evaluated the influence of IH on the cell signaling system of the airway during the allergen-induced challenge in an animal model, especially through the MAPK (mitogen-activated protein kinase) pathway. The protein concentrations of p-ERK/ERK, p-JNK/JNK, p-p38/p38, and pMEK/MEK were significantly reduced in the allergen-induced+IH group, compared to the allergen-induced group (p-value < 0.05 as considered statistically significant). The number of eosinophils, neutrophils, macrophages, and lymphocytes in the bronchoalveolar lavage fluid and Dp (Dermatophagoides pteronyssinus)-specific IgG2a and interleukins 4, 5, 13, and 17 were significantly reduced in the Dp+IH group, compared to the Dp group. These findings suggest that the MAPK pathway might be associated with the beneficial effect of IH on the attenuation of allergic response in an allergen-induced mouse model.     

Hyperoside Downregulates the Receptor for Advanced Glycation End Products (RAGE) and Promotes Proliferation in ECV304 Cells via the c-Jun N-Terminal Kinases (JNK) Pathway Following Stimulation by Advanced Glycation End-Products In Vitro

Hyperoside is a major active constituent in many medicinal plants which are traditionally used in Chinese medicines for their neuroprotective, anti-inflammatory and antioxidative effects. The molecular mechanisms underlying these effects are unknown. In this study, quiescent ECV304 cells were treated in vitro with advanced glycation end products (AGEs) in the presence or absence of hyperoside. The results demonstrated that AGEs induced c-Jun N-terminal kinases (JNK) activation and apoptosis in ECV304 cells. Hyperoside inhibited these effects and promoted ECV304 cell proliferation. Furthermore, hyperoside significantly inhibited RAGE expression in AGE-stimulated ECV304 cells, whereas knockdown of RAGE inhibited AGE-induced JNK activation. These results suggested that AGEs may promote JNK activation, leading to viability inhibition of ECV304 cells via the RAGE signaling pathway. These effects could be inhibited by hyperoside. Our findings suggest a novel role for hyperoside in the treatment and prevention of diabetes.     

ERK Inhibition Increases RANKL-Induced Osteoclast Differentiation in RAW 264.7 Cells by Stimulating AMPK Activation and RANK Expression and Inhibiting Anti-Osteoclastogenic Factor Expression

Bone absorption is necessary for the maintenance of bone homeostasis. An osteoclast (OC) is a monocyte–macrophage lineage cell that absorbs bone tissue. Extracellular signal-regulated kinases (ERKs) are known to play important roles in regulating OC growth and differentiation. In this study, we examined specific downstream signal pathways affected by ERK inhibition during OC differentiation. Our results showed that the ERK inhibitors PD98059 and U0126 increased receptor activator of NF-κB ligand (RANKL)-induced OC differentiation in RAW 264.7 cells, implying a negative role in OC differentiation. This is supported by the effect of ERK2-specific small interfering RNA on increasing OC differentiation. In contrast to our findings regarding the RAW 264.7 cells, the ERK inhibitors attenuated the differentiation of bone marrow-derived cells into OCs. The ERK inhibitors significantly increased the phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK) but not the activation of p38 MAPK, Lyn, and mTOR. In addition, while the ERK inhibition increased the expression of the RANKL receptor RANK, it decreased the expression of negative mediators of OC differentiation, such as interferon regulatory factor-8, B-cell lymphoma 6, and interferon-γ. These dichotomous effects of ERK inhibition suggest that while ERKs may play positive roles in bone marrow-derived cells, ERKs may also play negative regulatory roles in RAW 264.7 cells. These data provide important information for drug development utilizing ERK inhibitors in OC-related disease treatment.     

DYZ-2-90, a novel neo-tanshinlactone ring-opened compound, induces ERK-mediated mitotic arrest and subsequent apoptosis by activating JNK in human colorectal cancer cells

Over the past several decades, there has been a considerable and still growing interest in discovering natural products with anticancer potential from traditional Chinese medicine and increasing their anticancer selectivity by chemical modification. In addition, total synthesis of active compounds from natural products can overcome problems related to poor resource availability. DYZ-2-90 is a novel ring-opened compound modified from neo-tanshinlactone, which is isolated from Chinese medicinal herb tanshen. Both in vitro and in vivo tubulin polymerization assays showed that DYZ-2-90 directly bound to microtubules and rapidly induced tubulin depolymerization, inducing ERK-mediated mitotic arrest and subsequent apoptosis by JNK activation in cancer cells, respectively. These results suggest that the fate of cells that undergo mitotic arrest is dictated by two competing networks activated by DYZ-2-90: the cytoprotective ERK pathway and the stress-related JNK pathway. DYZ-2-90 is therefore a novel microtubule-destabilizing agent and a new drug candidate for cancer therapy. This paper provides a new insight into the model of mitotic cell death, which was proposed in order to elucidate how cancer cells respond to microtubule-interfering agents and prolonged cell cycle delay.     

Induction of Apoptosis by PQ1, a Gap Junction Enhancer that Upregulates Connexin 43 and Activates the MAPK Signaling Pathway in Mammary Carcinoma Cells

The mechanism of gap junction enhancer (PQ1) induced cytotoxicity is thought to be attributed to the change in connexin 43 (Cx43) expression; therefore, the effects of Cx43 modulation in cell survival were investigated in mammary carcinoma cells (FMC2u) derived from a malignant neoplasm of a female FVB/N-Tg(MMTV-PyVT)634Mul/J (PyVT) transgenic mouse. PQ1 was determined to have an IC₅₀ of 6.5 µM in FMC2u cells, while inducing an upregulation in Cx43 expression. The effects of Cx43 modulation in FMC2u cell survival was determined through transfection experiments with Cx43 cDNA, which induced an elevated level of protein expression similar to that seen with PQ1 exposure, or siRNA to silence Cx43 protein expression. Overexpression or silencing of Cx43 led to a reduction or an increase in cell viability, respectively. The mitogen-activated protein kinase (MAPK) family has been implicated in the regulation of cell survival and cell death; therefore, the gap junctional intercellular communication (GJIC)-independent function of PQ1 and Cx43 in the Raf/Mitogen-activated protein kinase/ERK kinase/extracellular-signal-regulated kinase (Raf-MEK-ERK) cascade of cellular survival and p38 MAPK-dependent pathway of apoptosis were explored. PQ1 treatment activated p44/42 MAPK, while the overexpression of Cx43 resulted in a reduced expression. This suggests that PQ1 affects the Raf-MEK-ERK cascade independent of Cx43 upregulation. Both overexpression of Cx43 and PQ1 treatment stimulated an increase in the phosphorylated form of p38-MAPK, reduced levels of the anti-apoptotic protein Bcl-2, and increased the cleavage of pro-caspase-3. Silencing of Cx43 protein expression led to a reduction in the phosphorylation of p38-MAPK and an increase in Bcl-2 expression. The mechanism behind PQ1-induced cytotoxicity in FMC2u mammary carcinoma cells is thought to be attributed to the change in Cx43 expression. Furthermore, PQ1-induced apoptosis through the upregulation of Cx43 may depend on p38 MAPK, highlighting that the effect of PQ1 on gap junctions as well as cellular survival via a MAPK-dependent pathway.     

Role of ERK Pathway in the Pathogenesis of Atopic Dermatitis and Its Potential as a Therapeutic Target

Atopic dermatitis (AD) is an eczematous skin disorder characterized by type 2 inflammation, barrier disruption, and intense itch. In addition to type 2 cytokines, many other cytokines, such as interferon gamma (IFN-γ), interleukin 17 (IL-17), and interleukin 22 (IL-22), play roles in the pathogenesis of AD. It has been reported that the extracellular signal-regulated kinase (ERK) is downstream of such cytokines. However, the involvement of the ERK pathway in the pathogenesis of AD has not yet been investigated. We examined the expression of p-ERK in mouse and human AD skin. We also investigated the effects of the topical application of an ERK inhibitor on the dermatitis score, transepidermal water loss (TEWL), histological change, and expression of filaggrin, using an AD-like NC/Nga murine model. The effects of an ERK inhibitor on filaggrin expression in normal human epidermal keratinocytes (NHEKs) and on chemokine production from bone marrow-derived dendritic cells (BMDCs) were also evaluated. p-ERK was highly expressed in mouse and human AD skin. Topical application of an ERK inhibitor alleviated the clinical symptoms, histological changes, TEWL, and decrease in expression of filaggrin in the AD-like NC/Nga murine model. The ERK inhibitor also restored the IL-4 induced reduction in the expression of filaggrin in NHEK, and inhibited chemokine production from BMDC induced by IL-4. These results indicate that the ERK pathway is involved in the pathogenesis of AD, and suggest that the ERK pathway has potential as a therapeutic target for AD in the future.     

Induction of Apoptosis by Matrine Derivative ZS17 in Human Hepatocellular Carcinoma BEL-7402 and HepG2 Cells through ROS-JNK-P53 Signalling Pathway Activation

Hepatocellular carcinoma (HCC) is one of the most common malignancies and ranks third among cancer-related deaths worldwide. Using matrine as a lead compound, 12 matrine derivatives were designed and synthesised, and their antiproliferative activities were evaluated in four cancer cell lines. Eight of the twelve compounds showed strong antiproliferative activity, with an IC₅₀ of <10 μM. The compound ZS17 exhibited strong antiproliferative activity in hepatocellular carcinoma cell lines with IC₅₀ values in the range of 3.014–3.388 μM, which was much lower than that of matrine. Furthermore, we explored the role of ZS17 in inducing apoptosis in HCC cells in vitro and in vivo, as well as possible mechanisms involved. ZS17 inhibited the proliferation of BEL-7402 and HepG2 cells in time- and dose-dependent manners. In addition, we found that ZS17 significantly induced apoptosis and ROS (reactive oxygen species) production, promoted JNK phosphorylation, activated p53, and activated the caspase signalling pathway. Furthermore, the antioxidant NAC, JNK inhibitor SP600125, and Si-JNK increased cell viability, re-established cell metastasis, and inhibited ZS17-induced apoptosis. An in vivo antitumour assay demonstrated that ZS17 significantly reduced the number of migrating HepG2 cells in zebrafish embryos and suppressed the growth of HepG2 xenografts in nude mice without any obvious side effects. Our study demonstrated that the ROS-JNK-P53 pathway plays an important role in the destruction of liver tumour cells by ZS17. Thus, ZS17 may represent a promising chemotherapeutic agent for the treatment of HCC patients.     

Characterization of the RAS/RAF/ERK Signal Cascade as a Novel Regulating Factor in Alpha-Amanitin-Induced Cytotoxicity in Huh-7 Cells

The well-known hepatotoxicity mechanism resulting from alpha-amanitin (α-AMA) exposure arises from RNA polymerase II (RNAP II) inhibition. RNAP Ⅱ inhibition occurs through the dysregulation of mRNA synthesis. However, the signaling pathways in hepatocytes that arise from α-AMA have not yet been fully elucidated. Here, we identified that the RAS/RAF/ERK signaling pathway was activated through quantitative phosphoproteomic and molecular biological analyses in Huh-7 cells. Bioinformatics analysis showed that α-AMA exposure increased protein phosphorylation in a time-dependent α-AMA exposure. In addition, phosphorylation increased not only the components of the ERK signaling pathway but also U2AF65 and SPF45, known splicing factors. Therefore, we propose a novel mechanism of α-AMA as follows. The RAS/RAF/ERK signaling pathway involved in aberrant splicing events is activated by α-AMA exposure followed by aberrant splicing events leading to cell death in Huh-7 cells.     

C Type Natriuretic Peptide Receptor Activation Inhibits Sodium Channel Activity in Human Aortic Endothelial Cells by Activating the Diacylglycerol-Protein Kinase C Pathway

The C-type natriuretic peptide receptor (NPRC) is expressed in many cell types and binds all natriuretic peptides with high affinity. Ligand binding results in the activation or inhibition of various intracellular signaling pathways. Although NPRC ligand binding has been shown to regulate various ion channels, the regulation of endothelial sodium channel (EnNaC) activity by NPRC activation has not been studied. The objective of this study was to investigate mechanisms of EnNaC regulation associated with NPRC activation in human aortic endothelial cells (hAoEC). EnNaC protein expression and activity was attenuated after treating hAoEC with the NPRC agonist cANF compared to vehicle, as demonstrated by Western blotting and patch clamping studies, respectively. NPRC knockdown studies using siRNA’s corroborated the specificity of EnNaC regulation by NPRC activation mediated by ligand binding. The concentration of multiple diacylglycerols (DAG) and the activity of protein kinase C (PKC) was augmented after treating hAoEC with cANF compared to vehicle, suggesting EnNaC activity is down-regulated upon NPRC ligand binding in a DAG-PKC dependent manner. The reciprocal cross-talk between NPRC activation and EnNaC inhibition represents a feedback mechanism that presumably is involved in the regulation of endothelial function and aortic stiffness.     

Suppression of LPS-Induced Inflammation and Cell Migration by Azelastine through Inhibition of JNK/NF-κB Pathway in BV2 Microglial Cells

The c-Jun N-terminal kinases (JNKs) are implicated in many neuropathological conditions, including neurodegenerative diseases. To explore potential JNK3 inhibitors from the U.S. Food and Drug Administration-approved drug library, we performed structure-based virtual screening and identified azelastine (Aze) as one of the candidates. NMR spectroscopy indicated its direct binding to the ATP-binding site of JNK3, validating our observations. Although the antihistamine effect of Aze is well documented, the involvement of the JNK pathway in its action remains to be elucidated. This study investigated the effects of Aze on lipopolysaccharide (LPS)-induced JNK phosphorylation, pro-inflammatory mediators, and cell migration in BV2 microglial cells. Aze was found to inhibit the LPS-induced phosphorylation of JNK and c-Jun. It also inhibited the LPS-induced production of pro-inflammatory mediators, including interleukin-6, tumor necrosis factor-α, and nitric oxide. Wound healing and transwell migration assays indicated that Aze attenuated LPS-induced BV2 cell migration. Furthermore, Aze inhibited LPS-induced IκB phosphorylation, thereby suppressing nuclear translocation of NF-κB. Collectively, our data demonstrate that Aze exerts anti-inflammatory and anti-migratory effects through inhibition of the JNK/NF-κB pathway in BV2 cells. Based on our findings, Aze may be a potential candidate for drug repurposing to mitigate neuroinflammation in various neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases.     

Iron Chelator Induces Apoptosis in Osteosarcoma Cells by Disrupting Intracellular Iron Homeostasis and Activating the MAPK Pathway

Osteosarcoma is a common malignant bone tumor in clinical orthopedics. Iron chelators have inhibitory effects on many cancers, but their effects and mechanisms in osteosarcoma are still uncertain. Our in vitro results show that deferoxamine (DFO) and deferasirox (DFX), two iron chelators, significantly inhibited the proliferation of osteosarcoma cells (MG-63, MNNG/HOS and K7M2). The viability of osteosarcoma cells was decreased by DFO and DFX in a concentration-dependent manner. DFO and DFX generated reactive oxygen species (ROS), altered iron metabolism and triggered apoptosis in osteosarcoma cells. Iron chelator-induced apoptosis was due to the activation of the MAPK signaling pathway, with increased phosphorylation levels of JNK, p38 and ERK, and ROS generation; in this process, the expression of C-caspase-3 and C-PARP increased. In an orthotopic osteosarcoma transplantation model, iron chelators (20 mg/kg every day, Ip, for 14 days) significantly inhibited the growth of the tumor. Immunohistochemical analysis showed that iron metabolism was altered, apoptosis was promoted, and malignant proliferation was reduced with iron chelators in the tumor tissues. In conclusion, we observed that iron chelators induced apoptosis in osteosarcoma by activating the ROS-related MAPK signaling pathway. Because iron is crucial for cell proliferation, iron chelators may provide a novel therapeutic strategy for osteosarcoma.     

The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior

Overactivation of the c-MET/HGF system is a feature of many cancers. We previously reported that type II testicular germ cell tumor (TGCT) cells express the c-MET receptor, forming non-seminomatous lesions that are more positive compared with seminomatous ones. Notably, we also demonstrated that NT2D1 non-seminomatous cells (derived from an embryonal carcinoma lesion) increase their proliferation, migration, and invasion in response to HGF. Herein, we report that HGF immunoreactivity is more evident in the microenvironment of embryonal carcinoma biopsies with respect to seminomatous ones, indicating a tumor-dependent modulation of the testicular niche. PI3K/AKT is one of the signaling pathways triggered by HGF through the c-MET activation cascade. Herein, we demonstrated that phospho-AKT increases in NT2D1 cells after HGF stimulation. Moreover, we found that this pathway is involved in HGF-dependent NT2D1 cell proliferation, migration, and invasion, since the co-administration of the PI3K inhibitor LY294002 together with HGF abrogates these responses. Notably, the inhibition of endogenous PI3K affects collective cell migration but does not influence proliferation or chemotactic activity. Surprisingly, LY294002 administered without the co-administration of HGF increases cell invasion at levels comparable to the HGF-administered samples. This paradoxical result highlights the role of the testicular microenvironment in the modulation of cellular responses and stimulates the study of the testicular secretome in cancer lesions.     

Human Interleukin 23 Receptor Induces Cell Apoptosis in Mammalian Cells by Intrinsic Mitochondrial Pathway Associated with the Down-Regulation of RAS/Mitogen-Activated Protein Kinase and Signal Transducers and Activators of Transcription factor 3 Signaling Pathways

The composition of IL-23R complex is similar to that of the IL-12 receptor (IL-12R) complex with a shared IL-12R-β1 chain. The IL-12R-β1 heterodimerizes with IL-23R and IL-12R-β2 to form IL-23R and IL-12R complexes, respectively. The IL-12R-β2 has been shown to function as a tumor suppressor gene and apoptotic inducer. However, whether IL-23R also functions in cell apoptosis is currently unknown. In this study, we demonstrate for the first time that overexpression of IL-23R markedly induces cell apoptosis in both 293ET and HeLa cells. The activations of caspase 3 and caspase 9 are induced by IL-23R. Mechanistic study reveals that IL-23R markedly inhibits RAS/MAPK and STAT3 but not STAT1 and PI-3K/Akt signaling pathways in both 293ET and HeLa cells. Overexpression of IL-23R significantly up-regulates IL-12Rβ1 expression but not IL-23α and IL-12β expressions in both cell lines. Therefore, our data strongly indicates that IL-23R is able to induce cell apoptosis by activating the intrinsic mitochondrial pathways associated with the inhibition in RAS/MAPK and STAT3 activations in mammalian cells.     

Involvement of the ERK/HIF-1α/EMT Pathway in XCL1-Induced Migration of MDA-MB-231 and SK-BR-3 Breast Cancer Cells

Chemokine–receptor interactions play multiple roles in cancer progression. It was reported that the overexpression of X-C motif chemokine receptor 1 (XCR1), a specific receptor for chemokine X-C motif chemokine ligand 1 (XCL1), stimulates the migration of MDA-MB-231 triple-negative breast cancer cells. However, the exact mechanisms of this process remain to be elucidated. Our study found that XCL1 treatment markedly enhanced MDA-MB-231 cell migration. Additionally, XCL1 treatment enhanced epithelial–mesenchymal transition (EMT) of MDA-MB-231 cells via E-cadherin downregulation and upregulation of N-cadherin and vimentin as well as increases in β-catenin nucleus translocation. Furthermore, XCL1 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, the effects of XCL1 on cell migration and intracellular signaling were negated by knockdown of XCR1 using siRNA, confirming XCR1-mediated actions. Treating MDA-MB-231 cells with U0126, a specific mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, blocked XCL1-induced HIF-1α accumulation and cell migration. The effect of XCL1 on cell migration was also evaluated in ER^-/HER2⁺ SK-BR-3 cells. XCL1 also promoted cell migration, EMT induction, HIF-1α accumulation, and ERK phosphorylation in SK-BR-3 cells. While XCL1 did not exhibit any significant impact on the matrix metalloproteinase (MMP)-2 and -9 expressions in MDA-MB-231 cells, it increased the expression of these enzymes in SK-BR-3 cells. Collectively, our results demonstrate that activation of the ERK/HIF-1α/EMT pathway is involved in the XCL1-induced migration of both MDA-MB-231 and SK-BR-3 breast cancer cells. Based on our findings, the XCL1–XCR1 interaction and its associated signaling molecules may serve as specific targets for the prevention of breast cancer cell migration and metastasis.     

G-Protein-Coupled Estrogen Receptor Expression in Rat Uterine Artery Is Increased by Pregnancy and Induces Dilation in a Ca2+ and ERK1/2 Dependent Manner

Increasing levels of estrogens across gestation are partly responsible for the physiological adaptations of the maternal vasculature to pregnancy. The G protein-coupled estrogen receptor (GPER) mediates acute vasorelaxing effects in the uterine vasculature, which may contribute to the regulation of uteroplacental blood flow. The aim of this study was to investigate whether GPER expression and vasorelaxation may occur following pregnancy. Elucidation of the functional signalling involved was also investigated. Radial uterine and third-order mesenteric arteries were isolated from non-pregnant (NP) and pregnant rats (P). GPER mRNA levels were determined and—concentration–response curve to the GPER-specific agonist, G1 (10⁻¹⁰–10⁻⁶ M), was assessed in arteries pre-constricted with phenylephrine. In uterine arteries, GPER mRNA expression was significantly increased and vasorelaxation to G1 was significantly enhanced in P compared with NP rats. Meanwhile, in mesenteric arteries, there was a similar order of magnitude in NP and P rats. Inhibition of L-type calcium channels and extracellular signal-regulated kinases 1/2 significantly reduced vasorelaxation triggered by G1 in uterine arteries. Increased GPER expression and GPER-mediated vasorelaxation are associated with the advancement of gestation in uterine arteries. The modulation of GPER is exclusive to uterine arteries, thus suggesting a physiological contribution of GPER toward the regulation of uteroplacental blood flow during pregnancy.