BACKGROUND: Enzalutamide, a second-generation androgen receptor (AR) pathway inhibitor, is widely used in the treatment of castration-resistant prostate cancer. However, after a period of enzalutamide treatment, patients inevitably develop drug resistance. In this study, we characterized leucine-rich repeated G-protein-coupled receptor 5 (LGR5) and explored its potential therapeutic value in prostate cancer. METHODS: A total of 142 pairs of tumor and adjacent formalin-fixed paraf-fin-embedded tissue samples from patients with prostate cancer were collected from the Pathology Department at Sun Yat-sen Memorial Hos-pital. LGR5 was screened by sequencing data of enzalutamide-resistant cell lines combined with sequencing data of lesions with different Gleason scores from the same patients. The biological function of LGR5 and its effect on enzalutamide resistance were investigated in vitro and in vivo . Glutathione-S-transferase (GST) pull-down, coimmunoprecipitation, Western blotting, and immunofluorescence assays were used to explore the specific binding mechanism of LGR5 and related pathway changes. RESULTS: LGR5 was significantly upregulated in prostate cancer and negatively correlated with poor patient prognosis. Overexpression of LGR5 promoted the malignant progression of prostate cancer and reduced sensitivity to enzalutamide in vitro and in vivo . LGR5 promoted the phosphorylation of glycogen synthase kinase-3β (GSK-3β) by binding heat shock protein 90,000 alpha B1 (HSP90AB1) and mediated the activation of the Wingless/integrated (WNT)/β-catenin signaling pathway. The increased β-catenin in the cytoplasm entered the nucleus and bound to the nuclear AR, promoting the transcription level of AR, which led to the enhanced tolerance of prostate cancer to enzalutamide. Reducing HSP90AB1 binding to LGR5 significantly enhanced sensitivity to enzalutamide. CONCLUSIONS LGR5 directly binds to HSP90AB1 and mediates GSK-3β phosphorylation, promoting AR expression by regulating the WNT/β-catenin signaling pathway, thereby conferring resistance to enzalutamide treatment in prostate cancer.
Male ; Humans ; Phenylthiohydantoin/pharmacology* ; Benzamides ; Receptors, G-Protein-Coupled/genetics* ; Nitriles ; Cell Line, Tumor ; HSP90 Heat-Shock Proteins/metabolism* ; Drug Resistance, Neoplasm/genetics* ; Prostatic Neoplasms/drug therapy* ; beta Catenin/metabolism* ; Receptors, Androgen/genetics* ; Animals ; Mice ; Wnt Signaling Pathway/physiology*

Skin, as the body's largest organ, acts as the primary defense mechanism against infection and injury. The maintenance of skin health heavily relies on the regulation of epidermal stem cells, crucial for ensuring epidermal homeostasis, hair regeneration, and the repair of epidermal injuries. Recent studies have placed a growing emphasis on G protein-coupled receptor (GPCR) in the context of understanding epidermal stem cells, uncovering its significant role in determining their fate. The activation of GPCR triggers the subsequent dissociation of the βγ subunits from the α subunit of G protein, leading to the modulation of various downstream signaling pathways, such as the WNT-BMP signaling crosstalk and the Gαs-PKA signaling pathway. These pathways collectively influence the fate of epidermal stem cells. Consequently, targeted GPCR therapy has emerged as a promising strategy for improving skin health by orchestrating the fate of epidermal stem cells, unveiling potential therapeutic targets that demand further investigation.
Humans ; Stem Cells/cytology* ; Receptors, G-Protein-Coupled/physiology* ; Animals ; Epidermal Cells/physiology* ; Signal Transduction ; Epidermis/physiology* ; Skin/cytology*

Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)-a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.
Endocytosis/physiology* ; Histatins/pharmacology* ; Humans ; Membrane Proteins ; Mitochondria/metabolism* ; RNA, Small Interfering/pharmacology* ; Receptors, G-Protein-Coupled/metabolism* ; Receptors, sigma

Over the past decade, there has been increasing attention on the interaction between microbiota and bile acid metabolism. Bile acids are not only involved in the metabolism of nutrients, but are also important in signal transduction for the regulation of host physiological activities. Microbial-regulated bile acid metabolism has been proven to affect many diseases, but there have not been many studies of disease regulation by microbial receptor signaling pathways. This review considers findings of recent research on the core roles of farnesoid X receptor (FXR), G protein-coupled bile acid receptor (TGR5), and vitamin D receptor (VDR) signaling pathways in microbial-host interactions in health and disease. Studying the relationship between these pathways can help us understand the pathogenesis of human diseases, and lead to new solutions for their treatments.
Bile Acids and Salts/metabolism* ; Gastrointestinal Microbiome ; Humans ; Inflammation/metabolism* ; Metabolic Syndrome/metabolism* ; Receptors, Calcitriol/physiology* ; Receptors, Cytoplasmic and Nuclear/physiology* ; Receptors, G-Protein-Coupled/physiology* ; Signal Transduction/physiology*

The recent development of tools to decipher the intricacies of neural networks has improved our understanding of brain function. Optogenetics allows one to assess the direct outcome of activating a genetically-distinct population of neurons. Neurons are tagged with light-sensitive channels followed by photo-activation with an appropriate wavelength of light to functionally activate or silence them, resulting in quantifiable changes in the periphery. Capturing and manipulating activated neuron ensembles, is a recently-designed technique to permanently label activated neurons responsible for a physiological function and manipulate them. On the other hand, neurons can be transfected with genetically-encoded Ca indicators to capture the interplay between them that modulates autonomic end-points or somatic behavior. These techniques work with millisecond temporal precision. In addition, neurons can be manipulated chronically to simulate physiological aberrations by transfecting designer G-protein-coupled receptors exclusively activated by designer drugs. In this review, we elaborate on the fundamental concepts and applications of these techniques in research.
Animals ; Autonomic Pathways ; physiology ; Calcium Signaling ; physiology ; Humans ; Nerve Net ; physiology ; Neurons ; physiology ; Optogenetics ; methods ; Receptors, G-Protein-Coupled ; physiology

Rodent MrgC receptor (Mas-related G-protein-coupled receptor subtype C) shares 65% sequence homology and similarities in terms of expression pattern and binding profile with human Mas-related gene X receptor 1 (hMrgX1). Therefore, researchers generally explore the role of hMrgX1 by studying the function of MrgC receptor. Murine MrgC receptor is uniquely expressed in small-diameter neurons of dorsal root ganglia (DRG) and trigeminal ganglia (TG), which is closely related to the transmission process of pain. This review summarizes the analgesic effects of intrathecal activation of MrgC receptors in pathological pain and morphine tolerance.
Animals ; Drug Tolerance ; Ganglia, Spinal ; Humans ; Mice ; Morphine ; pharmacology ; Pain ; Peptide Fragments ; Rats ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled ; physiology ; Trigeminal Ganglion

The aim of this study was to investigate whether G protein-coupled estrogen receptor (GPER) could alleviate hippocampal neuron injury under cerebral ischemia-reperfusion injury (CIRI) by acting on endoplasmic reticulum stress (ERS). The CIRI animal model was established by middle cerebral artery occlusion (MCAO). Female ovariectomized (OVX) Sprague-Dawley (SD) female rats were randomly divided into 4 groups: control, ischemia-reperfusion injury (MCAO), vehicle (MCAO+DMSO), and GPER-specific agonist G1 (MCAO+G1) groups. The neurobehavioral score was assessed by the Longa score method, the morphological changes of the neurons were observed by the Nissl staining, the cerebral infarction was detected by the TTC staining, and the neural apoptosis in the hippocampal CA1 region was detected by TUNEL staining. The distribution and expression of GRP78 (78 kDa glucose-regulated protein 78) in the hippocampal CA1 region were observed by immunofluorescent staining. The protein expression levels of GRP78, Caspase-12, CHOP and Caspase-3 were detected by Western blot, and the mRNA expression levels of GRP78, Caspase-12, and CHOP were detected by the real-time PCR. The results showed that the neurobehavioral score, cerebral infarct volume, cellular apoptosis index, as well as GRP78, Caspase-12 and CHOP protein and mRNA expression levels in the MCAO group were significantly higher than those of control group. And G1 reversed the above-mentioned changes in the MCAO+G1 group. These results suggest that the activation of GPER can decrease the apoptosis of hippocampal neurons and relieve CIRI, and its mechanism may involve the inhibition of ERS.
Animals ; Apoptosis ; Brain Ischemia ; CA1 Region, Hippocampal ; cytology ; Caspase 12 ; metabolism ; Caspase 3 ; metabolism ; Endoplasmic Reticulum Stress ; Female ; Heat-Shock Proteins ; metabolism ; Neurons ; cytology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Estrogen ; physiology ; Receptors, G-Protein-Coupled ; agonists ; Reperfusion Injury ; Transcription Factor CHOP ; metabolism

To investigate changes in the angiotensin converting enzyme 2 (ACE2) and angiotensin (1-7) [Ang (1-7)] and to explore the role of ACE2-Ang (1-7)-Mas receptor axis in hypertension with heart failure with preserved ejection fraction (HFPEF).
 Methods: A total of 70 patients with primary hypertension and preserved left ventricular ejection fraction (LVEF>50%) were recruited and patients were divided into a hypertension group (HBP) and a heart failure with preserved ejection fraction group (HFpEF) according to the diagnostic criteria of HFpEF. Thirty-five healthy participants were selected randomly as a control group. Enzyme linked immunosorbent assays (ELISA) method was used to detect concentration of Ang (1-7), ACE2, angiotensin II (Ang II), brain natriuretic peptide (BNP) in plasma. Male Sprague- Dawley (SD) rats was randomly divided into 2 groups: An HFpEF group (n=16) and a sham group (n=8). Rats (n=8) in the AAC group were given Ang (1-7) [0.5 mg/(kg.d), intraperitoneally] for 6 weeks, and the rest were given equal dose normal saline. Then all the rats were killed, and the hearts were taken out for hematoxylineosin (HE) staining. The protein expressions of angiotensin converting enzyme (ACE), ACE2, and Mas receptor were detected by Western blot.
 Results: The BNP and Ang II were significantly increased in the HBP group and the HFpEF group compared with the control group (P<0.01). There were not significantly different in levels of ACE2 and Ang (1-7) between the HBP group and control group (P>0.05), whereas those levels were significantly increased in the HFpEF group compared with the HBP group and control group (P<0.01). HE staining showed obvious hypertrophy of myocardial cell in the AAC group compared with the sham group. Hypertrophy of myocardial cell in the AAC+Ang (1-7) group was significantly higher than that in the AAC group. Expressions of ACE, ACE2, and Mas receptor proteins were significantly higher in the AAC group than those in the sham group (P<0.05), while the expressions of ACE2 and Mas receptor proteins in the AAC+Ang (1-7) group were significantly higher than those in the AAC group (P<0.05). There was no significant difference in the ACE protein expression between groups (P>0.05).
 Conclusion: ACE2 and Ang (1-7) are important predictive factors for the severity of heart failure and myocardial remodeling of HFpEF with hypertension; ACE2-Ang (1-7)-Mas receptor axis may play a protective role in preventing myocardial remodeling in HFpEF with hypertension.
Angiotensin I ; physiology ; Angiotensin II ; Animals ; Atrial Remodeling ; physiology ; Case-Control Studies ; Enzyme-Linked Immunosorbent Assay ; Heart Failure ; metabolism ; physiopathology ; Humans ; Hypertension ; metabolism ; physiopathology ; Male ; Peptide Fragments ; physiology ; Peptidyl-Dipeptidase A ; physiology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled ; physiology ; Stroke Volume ; Ventricular Function, Left ; physiology ; Ventricular Remodeling ; physiology

Objective: To investigate the expression of the G-protein coupled estrogen receptor (GPER) in the testis of the male mouse with kidney yin or kidney yang deficiency and its influence on the reproductive function of the mouse. METHODS: We randomized 30 six-week-old male Kunming mice into three groups of equal number: kidney yang deficiency, kidney yin deficiency, and normal control, and established the models of kidney yang deficiency and kidney yin deficiency by peritoneal injection of hydrocortisone at 50 mg/kg for 5 days and 25 mg/kg for 10 days, respectively. We observed the behavioral changes of the mice using the elevated plus-maze, exhaustive swimming and field experiment, examined the semen quality with the automatic sperm quality analyzer, calculated the average number of the offspring, measured the serum testosterone (T) and estradiol (E2) levels and T/E2 ratio by Roche electrochemiluminescence assay, and determined the localization and expression of GPER in the testis by immunohistochemistry and immunofluorescence staining. RESULTS: Compared with the mice with kidney yin deficiency, those with kidney yang deficiency showed remarkably fewer entries into the open arm and central area (P <0.05) and shorter time of exhaustive swimming (P <0.05), but no statistically significant difference in the time spent in the open arm or the central area (P >0.05); the latter group also exhibited significant decreases in the epididymal sperm count (［7.27 ± 1.30］ vs ［3.05 ± 1.06］ ×108/g, P <0.01), sperm motility (［54.15 ± 13.52］ vs ［51.57 ± 8.75］ %, P <0.01) and average number of the offspring (6.46 vs 4.33, P <0.05), a slight increase in the rate of morphologically abnormal sperm (［13.42 ± 2.32］ vs ［15.39 ± 2.48］ %, P >0.05), and markedly reduced serum T (［24.96 ± 6.18］ vs ［16.72 ± 5.92］ ng/dl,P <0.05), E2 (［19.81 ± 4.01］ vs ［15.24 ± 1.11］ pg/ml,P <0.05) and T/E2 ratio (1.41 vs 1.25, P <0.05). The expression of GPER was found in the cytoplasm of the Leydig cells, negative in the nuclei and cell membrane, significantly higher in the kidney yang than in the kidney yin deficiency group (P <0.05). CONCLUSIONS The numbers of sperm and offspring decreased while the percentage of morphologically abnormal sperm increased in both the kidney yang and kidney yin deficiency mice, even more significantly in the former, which might be associated with the up-regulated expression of GPER in the testis of the mouse with kidney yang deficiency and consequently the reduced serum T level and T/E2 ratio.
Animals ; Drugs, Chinese Herbal ; Kidney Diseases ; metabolism ; Male ; Mice ; Random Allocation ; Receptors, Estrogen ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Reproduction ; physiology ; Semen Analysis ; Testis ; metabolism ; Yang Deficiency ; metabolism ; Yin Deficiency ; metabolism

OBJECTIVETo predict and identify the target gene of miR-145, and to explore the underlying mechanism of the inhibition of miR-145 on drug resistance to Oxaliplatin (L-OHP) in human colorectal cancer cells.

METHODSL-OHP-resistant human colorectal cancer cell line (HCT116/L-OHP) was established in vitro by exposing to increased concentrations of L-OHP in cell culture medium. MiR-145-mimics and its negative control (NC-miRNA) were transfected into HCT116/L-OHP cells using liposome to establish HCT116/L-OHPover-expressing miR-145 and HCT116/L-OHP. The target genes of miR-145 were predicted by bioinformatic analysis, and validated by dual luciferase activity assay. After determination of G protein coupled receptor 98(GPR98) as target gene, corresponding plasmids were constructed and transfected to establish HCT116/L-OHPover-expressing GPR98 and HCT116/L-OHP. HCT116/L-OHP cells over-expressing both GPR98 and miR-145 (HCT116/L-OHP) were acquired through modification of the binding sites of GPR98 cDNA with miR-145. CCK-8 assay was used to assess the proliferation (A value) and sensitivity to L-OHP (the lower the IC50, the stronger the sensitivity) in HCT116/L-OHP cells. Real-time quantitative PCR was used to measure the mRNA expression of miR-145 and GPR98. Western blot was used to examine the protein expression of GPR98 and drug-resistant associated protein, such as P-glycoprotein (gp), multiple drug-resistance protein 1(MRP1), cancer-inhibition gene PTEN.

RESULTSHCT116/L-OHP cell line was successfully established with ICof (42.34±1.05) mg/L and miR-145 mRNA expression of 0.27±0.04, which was higher than (9.81±0.95) mg/L (t=39.784, P=0.000) and lower than 1.00±0.09 (t=13.021, P=0.000) in HCT116 cells. Based on HCT116/L-OHP cells, HCT116/L-OHPcells were established successfully, with relative miR-145 expression of 10.01±1.05, which was higher than 1.06±0.14 in HCT116/L-OHPand 1.00±0.16 in HCT116/L-OHP (F=161.797, P=0.000). GPR98 was identified to be the target gene of miR-145. The relative mRNA and protein expressions of GPR98 in HCT116/L-OHPcells were 8.48±0.46 and 1.71±0.09, respectively, which were higher than those in HCT116/L-OHP(mRNA: 3.65±0.40, protein: 1.21±0.10) and HCT116/L-OHP (mRNA: 3.49±0.35, protein: 1.22±0.08; all P<0.05). The A value was 1.31±0.10, and the relative protein expressions of P-gp and MRP1 were 1.53±0.18 and 1.49±0.20 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP (A value: 0.82±0.08, relative protein expression: 1.00±0.06 and 1.21±0.13, all P<0.05). The A value was 0.89±0.08, and the relative protein expressions of P-gp and MRP were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP(A value: 0.20±0.05, relative protein expression: 0.20±0.07, 0.55±0.10, all P<0.05). The relative protein expression of PTEN in HCT116/L-OHPcells was 0.12±0.03, which was lower than 1.25±0.14 in HCT116/L-OHP cells(P<0.05). In addition, relative protein expressions of P-gp and MRP1 were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHPcells (0.20±0.07 and 0.55±0.10), while PTEN expression in HCT116/L-OHPcells was lower as compared to HCT116/L-OHPcells (1.41±0.16 vs. 1.98±0.13, P<0.05).

CONCLUSIONMiR-145 inhibits drug resistance to L-OHP of HCT116 cells through suppressing the expression of target gene GPR98.

ATP Binding Cassette Transporter, Sub-Family B ; drug effects ; ATP-Binding Cassette, Sub-Family B, Member 1 ; drug effects ; Cell Line, Tumor ; drug effects ; physiology ; Colorectal Neoplasms ; physiopathology ; Down-Regulation ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; physiology ; HCT116 Cells ; drug effects ; physiology ; Humans ; In Vitro Techniques ; MicroRNAs ; genetics ; pharmacology ; Multidrug Resistance-Associated Proteins ; drug effects ; Organoplatinum Compounds ; pharmacology ; PTEN Phosphohydrolase ; drug effects ; RNA, Messenger ; Receptors, G-Protein-Coupled ; drug effects ; genetics