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Background Ethanol (EtOH) exposure throughout gestation and breastfeeding leads to multiple adverse outcomes in the hepatic system. Under oxidative stress, alterations in the liver are related to the inhibition of induced nitric oxide synthase activity in sinusoidal cells as a consequence of low expression of G-protein-coupled receptor (GPCR)-kinase interacting (GIT1). Here, we hypothesized that both glutathione peroxidase 1 (GPx1) and GIT1 could be altered by EtOH exposure during the third trimester of human equivalent development. Methods We exposed rats during the third trimester equivalent [postnatal days (PD) 2-8] to moderate levels of maternal EtOH (20%). GPx1 and GIT1 expression was detected by western blotting, and the antioxidant activity of glutathione peroxidase GPx and the concentration of hepatic carbonyl groups (CG were determined by spectrophotometry. Serum biochemistry parameters such as alanine aminotransferase (ALT), glucose (gluc), cholesterol (chol), and triglycerides (TG) were also measured. Results We found that ethanol decreased both GIT1 and GPx1 selenoprotein expression, affecting GPx antioxidant activity and increasing protein oxidation. Conclusions These results demonstrate for the first time that the GPx antioxidant system altered by EtOH exposure during the third trimester of development is related to a parallel decrease in GIT1 expression [1].

Spinal cord ischemia-reperfusion injury (SCIRI) is a major contributor to neurological damage and mortality associated with spinal cord dysfunction. This study aims to explore the possible mechanism of Propofol and G-protein-coupled receptorinteracting protein 1 (GIT1) in regulating SCIRI in rat models. SCIRI rat models were established and injected with Propofol, over expression of GIT1 (OE-GIT1), or PI3K inhibitor (LY294002). The neurological function was assessed using Tarlov scoring system, and Hematoxylin & Eosin (H&E) staining was applied to observe morphology changes in spinal cord tissues. Cell apoptosis, blood-spinal cord barriers (BSCB) permeability, and inflammatory cytokines were determined by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, evans blue (EB) staining, and enzyme-linked immuno sorbent assay (ELISA), respectively. Reverse transcription-quantitative polymerase chain reaction and western blot were used to detect the expression levels of GIT1, endothelial nitric oxide synthase (eNOS), PI3K/AKT signal pathway and apoptosis-related proteins. SCIRI rats had decreased expressions of GIT1 and PI3K/AKT-related proteins, whose expressions can be elevated in response to Propofol treatment. LY294002 can also decrease GIT1 expression levels in SCIRI rats. Propofol can attenuate neurological dysfunction induced by SCIRI, decrease spinal cord tissue injury and BSCB permeability in addition to suppressing cell apoptosis and inflammatory cytokines, whereas further treatment by LY294002 can partially reverse the protective effect of Propofol on SCIRI. Propofol can activate PI3K/AKT signal pathway to increase GIT1 expression level, thus attenuating SCIRI in rat models. [ABSTRACT FROM AUTHOR]

Chenkun Wang,1,2,* Jing Gu,2,* Hongwei Li,2,* Bo Zhao,2 Tao Yu,1,2 Chun-Ling Guo,2 Mouxin Huang,2 Weiwei Jiang,1 Qin Ouyang2 1Department of Pharmacy, the Second Affiliated Hospital, Chongqing Medical University, Chongqing, People’s Republic of China; 2Department of Medicinal Chemistry, College of Pharmacy, Third Military Medical University, Chongqing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Weiwei Jiang, Department of Pharmacy, the Second Affiliated Hospital, Chongqing Medical University, No. 1 Yixueyuan Road, Yuzhong District, Chongqing, 400010, People’s Republic of China, Email 304035@cqmu.edu.cn Qin Ouyang, Department of Medicinal Chemistry, College of Pharmacy, Third Military Medical University, No. 30 Gaotanyan St., Shapingba District, Chongqing, 400038, People’s Republic of China, Email ouyangq@tmmu.edu.cnBackground and Objective: GIT1 (G-protein-coupled receptor kinase interacting protein-1) has been found to be highly related with cancer cell invasion and metastasis in many cancer types. β-Pix (p21-activated kinase-interacting exchange factor) is one of the proteins that interact with GIT1. Targeting GIT1/β-Pix complex might be a potential therapeutic strategy for interfering cancer metastasis. However, at present, no well-recognized small-molecule inhibitor targeting GIT1/β-Pix is available. Thus, we aim to discover novel GIT1/β-Pix inhibitors with simple scaffold, high activity and low toxicity to develop new therapeutic strategies to restrain cancer metastasis.Methods: GIT1/β-Pix inhibitors were identified from ChemBridge by virtual screening. Briefly, the modeling of GIT1 was performed and the establishment of GIT1/β-Pix binding pocket enabled the virtual screening to identify the inhibitor. In addition, direct binding of the candidate molecules to GIT1 was detected by biolayer interferometry (BLI) to discover the hit compound. Furthermore, the inhibitory effect on invasion of stomach and colon cancer cells in vitro was carried out by the transwell assay and detection of epithelial-mesenchymal transition (EMT)-related proteins. Finally, the binding mode of hit compound to GIT1 was estimated by molecular dynamics simulation to analyze the key amino residues to guide further optimization.Results: We selected the top 50 compounds from the ChemBridge library by virtual screening. Then, by skeleton similarity analysis nine compounds were selected for further study. Furthermore, the direct interaction of nine compounds to GIT1 was detected by BLI to obtain the best affinitive compound. Finally, 17302836 was successfully identified (KD = 84.1± 2.0 μM). In vitro tests on 17302836 showed significant anti-invasion effect on gastric cancer and colorectal cancer.Conclusion: We discovered a new GIT1/β-Pix inhibitor (17302836) against gastrointestinal cancer invasion and metastasis. This study provides a promising candidate for developing new GIT1/β-Pix inhibitors for tumor treatment.Keywords: hit discovery, virtual screening, GIT1, β-Pix, gastrointestinal cancers, invasion

Background Ethanol (EtOH) exposure throughout gestation and breastfeeding leads to multiple adverse outcomes in the hepatic system. Under oxidative stress, alterations in the liver are related to the inhibition of induced nitric oxide synthase activity in sinusoidal cells as a consequence of low expression of G-protein-coupled receptor (GPCR)-kinase interacting (GIT1). Here, we hypothesized that both glutathione peroxidase 1 (GPx1) and GIT1 could be altered by EtOH exposure during the third trimester of human equivalent development. Methods We exposed rats during the third trimester equivalent [postnatal days (PD) 2-8] to moderate levels of maternal EtOH (20%). GPx1 and GIT1 expression was detected by western blotting, and the antioxidant activity of glutathione peroxidase GPx and the concentration of hepatic carbonyl groups (CG were determined by spectrophotometry. Serum biochemistry parameters such as alanine aminotransferase (ALT), glucose (gluc), cholesterol (chol), and triglycerides (TG) were also measured. Results We found that ethanol decreased both GIT1 and GPx1 selenoprotein expression, affecting GPx antioxidant activity and increasing protein oxidation. Conclusions These results demonstrate for the first time that the GPx antioxidant system altered by EtOH exposure during the third trimester of development is related to a parallel decrease in GIT1 expression [1].

Objective To investigate the influence of circ_BACH2 on the malignant biological behavior of papillary thyroid cancer and its molecular mechanism. Methods Cancer tissues and paracancer tissues of 51 patients with papillary thyroid carcinoma from the Fourth Central Hospital of Tianjin between 2017 and 2019 were collected. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expressions of circ_BACH2, miR-370-3p and G protein coupled receptor kinase interacting factor 1 (GIT1) mRNA in tissues and cells; flow cytometry to detect cell apoptosis and cell cycle; plate clone formation experiment to detect the number of cell clones; cell counting kit 8 (CCK-8) to detect cell proliferation; Transwell array to detect cell migration and invasion; western blot to detect protein expressions; dual luciferase report experiment to detect the targeting relationship between circ_BACH2, miR-370-3p and GIT1; the nude mouse tumor formation experiment to detect the effect of circ_BACH2 on tumors in mice. Results Compared with adjacent tissues, the expressions of circ_BACH2 and GIT1 in papillary thyroid cancer tissues was increased, while the expression of miR-370-3p was decreased. Compared with Nthy-ori3-1 cells, the expressions of circ_BACH2 in papillary thyroid cancer cells TPC-1 and SW579 were increased, the mRNA and protein levels of GIT1 were increased, miR-370-3p expression was decreased. The expression level of GIT1 mRNA was negatively correlated with that of miR-370-3p (r=−0.634), and the expression level of circ_BACH2 was positively correlated with that of GIT1 (r=0.635). The expression level of circ_BACH2 was negatively correlated with that of miR-370-3p (r= − 0.394, P＜0.05). Circ_BACH2 and miR-370-3p has a binding site at the 3' UTR of GIT1. After knocking down circ_BACH2, the proportion of G0/G1 cells in papillary thyroid cancer cells TPC-1 and SW579 was increased, the proportion of S-phase cells was decreased and the proportion of G2/M-phase cells did not change significantly. The cell absorbance value was lower than that in si-NC group. The number of cell clone formation was decreased (43±5 vs 100±6, 54±8 vs 100±9); the cell apoptosis rate was increased [(19.60±2.40)% vs (4.30±0.20)%, (18.10±2.10)% vs (5.10±0.23)% ]; cell migration number was decreased (61±7 vs 134±15, 58±6 vs 112±11), the invasion number was also decreased (45±6 vs 113±11, 47±4 vs 92±9); the expressions of Snail and Twist1 were decreased, and the expression of E-cadherin was increased (P＜0.000). Inhibition of miR-370-3p expression reversed the effect of circ_BACH2 knockdown on proliferation, migration, invasion and apoptosis of thyroid papillary cancer cells. Overexpression of GIT1 reversed the effects of overexpression of miR-370-3p on proliferation, migration, invasion and apoptosis of thyroid papillary cancer cells. Mice injected with TPC-1 cells stably transfected with sh-circ_BACH2 showed a reduction in tumor volume [(535±91) mm³ vs (857±114) mm³] after 35 days of culture; tumor weight was decreased [(0.62±0.13) mg vs (1.06±0.15) mg, P＜0.05]; the expressions of circ_BACH2 and GIT1 were decreased, and the expression of miR-370-3p was increased in nude mouse tumor tissue. Conclusion Silencing circ_BACH2 may inhibit the proliferation, migration and invasion of papillary thyroid cancer cells in vitro, promote cell apoptosis, and inhibit tumor growth in vivo through targeted regulation of miR-370-3p/GIT1. [ABSTRACT FROM AUTHOR]

Bone defects have long been a major healthcare issue because of the difficulties in regenerating bone mass volume and the high cost of treatment. G protein-coupled receptor kinase 2 interacting protein 1 (GIT1) has been proven to play an important role both in vascular development and in bone fracture healing. In this study, a type of thermoresponsive injectable hydrogel from oligoethylene glycol-based dendronized chitosan (G1-CS) was loaded with GIT1-plasmids (G1-CS/GIT1), and used to fill unicortical bone defects. RT-PCR analysis confirmed that G1-CS/GIT1 enhanced DNA transfection in MSCs both in vitro and in vivo. From the results of micro-CT, RT-PCR and histological analysis, it can be concluded that G1-CS/GIT1 accelerated the bone healing rate and increased the amount of neovascularization around the bone defects. In addition, an adeno-associated virus (AAV)-GIT1 was constructed to transfect mesenchymal stem cells. The results of capillary tube formation assay, immunofluorescence staining and western blot analysis proved that high expression of GIT1 induces mesenchymal stem cells to differentiate into endothelial cells. RT-PCR analysis and capillary tube formation assay confirmed that the Notch signaling pathway was activated in the differentiation process. Overall, we developed an efficient strategy through combination of injectable hydrogel and G1T1 for bone tissue engineering.

Background. Despite G-protein-coupled receptor kinase-interacting protein-1 (GIT1) being recognized as a new promoter gene in some types of cancer, its effect on human pan-cancers and liver hepatocellular carcinoma (LIHC) remains unclear. Objectives. To elucidate the molecular mechanisms of GIT1 in pan-cancer and LIHC. Materials and methods. Various bioinformatics approaches were utilized to elucidate the oncogenic effects of GIT1 on human pan-cancers. Results. The GIT1 was aberrantly expressed in pan-cancers and associated with the clinical stage. Moreover, the upregulation of GIT1 expression was indicative of poor overall survival (OS) in patients with LIHC, skin cutaneous melanoma (SKCM) and uterine corpus endometrial carcinoma (UCEC), as well as of poor disease-free survival (DFS) in patients with LIHC and UCEC. Furthermore, GIT1 levels were correlated with cancer-associated fibroblasts (CAFs) in adrenocortical carcinoma (ACC), cervical squamous cell carcinoma (CESC) and LIHC. The analysis of single-cell sequencing data revealed an association of GIT1 levels with apoptosis, cell cycle and DNA damage. In addition, multivariate Cox analysis indicated that high GIT1 levels were an independent risk factor for shorter OS in patients with LIHC. Finally, the gene set enrichment analysis revealed INFLAMMATORY_RESPONSE pathway and IL2_STAT5_SIGNALING to be the most enriched in LIHC. Conclusions. Our data demonstrate the oncogenic effects of GIT1 on various cancers. We believe that GIT1 can serve as a biomarker for LIHC. [ABSTRACT FROM AUTHOR]

ADHD is a typical neurodevelopmental disorder with a high prevalence rate. NSCs in the subventricular zone (SVZ) are closely related to neurodevelopmental disorder and can affect olfactory function by neurogenesis and migratory route. Although olfactory dysfunction is one of the symptoms of ADHD, the relevance of cells in the olfactory bulb derived from NSCs has not been studied. Therefore, we investigated olfactory memory and NSCs in Git1-deficient mice, under the ADHD model. Interestingly, only adult male G protein-coupled receptor kinase-interacting protein-1 (GIT1)-deficient (+/−, HE) mice showed impaired olfactory memory, suggesting sex and age dependence. We performed adult NSCs culture from the SVZ and observed distinct cell population in both sex and genotype. Taken together, our study suggests that the altered differentiation of NSCs in GIT1+/− mice can contribute to olfactory dysfunction in ADHD. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of miR-493-5p on proliferation, invasion and migration of ovarian cancer cells and underlying mechanism. Methods Culture human normal ovarian epithelial cell lines IOSE80 and ovarian cancer cell lines A2780, SKOV3, OVCAR3, transfected the miR-493-5p over-expression vectors, miR-493-5p suppressed expression vectors and G protein coupled receptor kinase interacting factor 1 (GIT1) over-expression vectors into SKOV3 cells; RT-qPCR was used to detect miR-493-5p and GIT1 mRNA expression; Western blot was used to detect protein expression; MTT assay was used to detect cell viability; Transwell was used to detect cell migration and invasion; dual luciferase report experiment was applied for detection of the targeting relationship between miR-493-5p and GIT1. Results In ovarian cancer cells A2780, SKOV3, OVCAR3, miR-493-5p were weakly expressed, and GIT1 was highly expressed (P＜0.05); After over-expression of miR-493-5p, the proliferation activity, migration and invasion number of ovarian cancer cell SKOV3 were decreased (P＜0.05). miR-493-5p targeted at the regulation of GIT1 expression, and over-expression of GIT1 reduced the inhibitory effect of miR- 493-5p on proliferation, migration and invasion of ovarian cancer cell SKOV3 and expression of p-MEK1/2, p-ERK1/2, pc-Jun and pc-Fos. Conclusions miR-493-5p can inhibit the proliferation, migration and invasion of ovarian cancer cells SKOV3. The mechanism may be related to the targeted regulation of GIT1 and MEK1/2-ERK1/2 pathway, which thus provides some new targets for diagnosis, prevention and treatment for ovarian cancer.

Abstract Background UBTF is an HMGB-box DNA binding protein and a necessary Pol I/Pol II basal transcription factor. It has been found that UBTF involves in carcinogenesis and progression of a few cancers. Nevertheless, the the biological function and potential molecular mechanism of UBTF in melanoma are still not clear and need to be clarified. Methods UBTF and GIT1 expressions in melanoma specimens and cell lines were examined by quantitative real-time PCR (qRT-PCR) and Western blot. MTT and colony formation assays were used to investigate the effects of UBTF and GIT1 on melanoma cell proliferation. Cell cycle and apoptosis assays were detected by flow cytometry. Tumor formation assay was used to analyze the effect of UBTF on melanoma growth. Bioinformatics predicting, chromatin immunoprecipitation (ChIP)-qRT-PCR and reporter gene assay were fulfilled for verifing GIT1 as UBTF targeting gene. Results Here we reported that UBTF mRNA and protein expressions were upregulated in primary melanoma specimens and cell lines. UBTF overexpression facilitated melanoma cell proliferation and cell cycle progression and restrained. Silencing UBTF suppressed cell multiplication, cell cycle progression and tumor growth, and promoted apoptosis. UBTF expression was positively related with GIT1 expression in human melanoma tissues. It was verified that UBTF promoted GIT1 transcription in melanoma cells through binding to the promoter region of GIT1. Furthermore, GIT1 overexpression promoted melanoma cell growth and suppressed apoptosis. Knockdown of GIT1 inhibited cell multiplication and induced apoptosis. Overexpression of GIT1 eliminated the effects of silencing UBTF on melanoma cells. Importantly, UBTF activated MEK1/2-ERK1/2 signalling pathways by upregulating GIT1 expression. Conclusions Our study demonstrates that UBTF promotes melanoma cell proliferation and cell cycle progression by promoting GIT1 transcription, thereby activating MEK1/2-ERK1/2 signalling pathways. The findings indicate that UBTF plays a crucial function in melanoma and may be a potential therapeutic target for the treatment of this disease.

Background and Purpose: The Dll4‐Notch1 signalling pathway plays an important role in sprouting angiogenesis, vascular remodelling and arterial or venous specificity. Genetic or pharmacological inhibition of Dll4‐Notch1 signalling leads to excessive sprouting angiogenesis. However, transcriptional inhibitors of Dll4‐Notch1 signalling have not been described. Experimental Approach We designed a new peptide targeting Notch signalling, referred to as TAT‐ANK, and assessed its effects on angiogenesis. In vitro, tube formation and fibrin gel bead assay were carried out, using human umbilical vein endothelial cells (HUVECs). In vivo, Matrigel plug angiogenesis assay, a developmental retinal model and tumour models in mice were used. The mechanisms underlying TAT‐ANK activity were investigated by immunochemistry, western blotting, immunoprecipitation, RT‐qPCR and luciferase reporter assays. Key Results: The amino acid residues 179–191 in the G‐protein‐coupled receptor‐kinase‐interacting protein‐1 (GIT1‐ankyrin domain) are crucial for GIT1 binding to the Notch transcription repressor, RBP‐J. We designed the peptide TAT‐ANK, based on residues 179–191 in GIT1. TAT‐ANK significantly inhibited Dll4 expression and Notch 1 activation in HUVECs by competing with activated Notch1 to bind to RBP‐J. The analyses of biological functions showed that TAT‐ANK promoted angiogenesis in vitro and in vivo by inhibiting Dll4‐Notch1 signalling. Conclusions and Implications: We synthesized and investigated the biological actions of TAT‐ANK peptide, a new inhibitor of Notch signalling. This peptide will be of significant interest to research on Dll4‐Notch1 signalling and to clinicians carrying out clinical trials using Notch signalling inhibitors. Furthermore, our findings will have important conceptual and therapeutic implications for angiogenesis‐related diseases. [ABSTRACT FROM AUTHOR]

Abstract Background A variety of DNA-based methods have been applied to identify genetic markers of attention deficit hyperactivity disorder (ADHD), but the connection to RNA-based gene expression has not been fully exploited. Methods Using well defined cohorts of discordant, monozygotic twins from the Michigan State University Twin Registry, and case-controlled ADHD cases in adolescents, the present studies utilized advanced single molecule RNA sequencing to identify expressed changes in whole blood RNA in ADHD. Multiple analytical strategies were employed to narrow differentially expressed RNA targets to a small set of potential biomarkers of ADHD. Results RNA markers common to both the discordant twin study and case-controlled subjects further narrowed the putative targets, some of which had been previously associated with ADHD at the DNA level. The potential role of several differentially expressed genes, including ABCB5, RGS2, GAK, GIT1 and 3 members of the galactose metabolism pathway (GALE, GALT, GALK1) are substantiated by prior associations to ADHD and by established mechanistic connections to molecular pathways relevant to ADHD and behavioral control. Conclusions The convergence of DNA, RNA, and metabolic data suggests these may be promising targets for diagnostics and therapeutics in ADHD.

The discovery of new biomarkers would be very valuable to improve the detection of early Alzheimer’s disease (AD). DNA methylation marks may serve as epigenetic biomarkers of early AD. Here we identified epigenetic marks that are present in the human hippocampus from the earliest stages of AD. A previous methylome dataset of the human AD hippocampus was used to select a set of eight differentially methylated positions (DMPs) since early AD stages. Next, bisulphite pyrosequencing was performed in an expanded homogeneous cohort of 18 pure controls and 35 hippocampal samples with neuropathological changes of pure AD. Correlation between DNA methylation levels in DMPs and phospho-tau protein burden assessed by immunohistochemistry in the hippocampus was also determined. We found four DMPs showing higher levels of DNA methylation at early AD stages compared to controls, involving ELOVL2, GIT1/TP53I13 and the histone gene locus at chromosome 6. DNA methylation levels assessed by bisulphite pyrosequencing correlated with phospho-tau protein burden for ELOVL2 and HIST1H3E/HIST1H3 F genes. In this discovery study, a set of four epigenetic marks of early AD stages have been identified in the human hippocampus. It would be worth studying in-depth the specific pathways related to these epigenetic marks. These early alterations in DNA methylation in the AD hippocampus could be regarded as candidate biomarkers to be explored in future translational studies. Abbreviations AD: Alzheimer’s disease; DMPs: Differentially methylated positions; CSF: Cerebrospinal fluid; βA42: β-amyloid 42; PET: positron emission tomography; 5mC: 5-methyl cytosine; CpG: cytosine-guanine dinucleotides; ANK1: ankyrin-1; BIN1: amphiphysin II; p-tau: hyperphosphorylated tau; CERAD: Consortium to Establish A Registry for Alzheimer’s Disease; SD: standard deviation; ANOVA: one-way analysis of variance; VLCFAs: very long-chain fatty acids; DHA: docosahexaenoic acid; mTOR: mechanistic target of rapamycin.

Spinal cord ischemia-reperfusion injury (SCIRI) is a major contributor to neurological damage and mortality associated with spinal cord dysfunction. This study aims to explore the possible mechanism of Propofol and G-protein-coupled receptor-interacting protein 1 (GIT1) in regulating SCIRI in rat models. SCIRI rat models were established and injected with Propofol, over expression of GIT1 (OE-GIT1), or PI3K inhibitor (LY294002). The neurological function was assessed using Tarlov scoring system, and Hematoxylin & Eosin (H&E) staining was applied to observe morphology changes in spinal cord tissues. Cell apoptosis, blood-spinal cord barriers (BSCB) permeability, and inflammatory cytokines were determined by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, evans blue (EB) staining, and enzyme-linked immuno sorbent assay (ELISA), respectively. Reverse transcription-quantitative polymerase chain reaction and western blot were used to detect the expression levels of GIT1, endothelial nitric oxide synthase (eNOS), PI3K/AKT signal pathway and apoptosis-related proteins. SCIRI rats had decreased expressions of GIT1 and PI3K/AKT-related proteins, whose expressions can be elevated in response to Propofol treatment. LY294002 can also decrease GIT1 expression levels in SCIRI rats. Propofol can attenuate neurological dysfunction induced by SCIRI, decrease spinal cord tissue injury and BSCB permeability in addition to suppressing cell apoptosis and inflammatory cytokines, whereas further treatment by LY294002 can partially reverse the protective effect of Propofol on SCIRI. Propofol can activate PI3K/AKT signal pathway to increase GIT1 expression level, thus attenuating SCIRI in rat models., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Spinal cord injury (SCI) can result in severe motor and sensory deficits, for which currently no effective cure exists. The pathological process underlying this injury is extremely complex and involves many cell types in the central nervous system. In this study, we have uncovered a novel function for macrophage G protein-coupled receptor kinase-interactor 1 (GIT1) in promoting remyelination and functional repair after SCI. Using GIT1 flox/flox Lyz2-Cre (GIT1 CKO) mice, we identified that GIT1 deficiency in macrophages led to an increased generation of tumor necrosis factor-alpha (TNFα), reduced proportion of mature oligodendrocytes (mOLs), impaired remyelination, and compromised functional recovery in vivo. These effects in GIT1 CKO mice were reversed with the administration of soluble TNF inhibitor. Moreover, bone marrow transplantation from GIT1 CWT mice reversed adverse outcomes in GIT1 CKO mice, further indicating the role of macrophage GIT1 in modulating spinal cord injury repair. Our in vitro experiments showed that macrophage GIT1 plays a critical role in secreting TNFα and influences the differentiation of oligodendrocyte precursor cells (OPCs) after stimulation with myelin debris. Collectively, our data uncovered a new role of macrophage GIT1 in regulating the transformation of OPCs into mOLs, essential for functional remyelination after SCI, suggesting that macrophage GIT1 could be a promising treatment target of SCI., (© 2024 Wiley Periodicals LLC.)

De novo protein synthesis is required for synapse modifications underlying stable memory encoding. Yet neurons are highly compartmentalized cells and how protein synthesis can be regulated at the synapse level is unknown. Here, we characterize neuronal signaling complexes formed by the postsynaptic scaffold GIT1, the mechanistic target of rapamycin (mTOR) kinase, and Raptor that couple synaptic stimuli to mTOR-dependent protein synthesis; and identify NMDA receptors containing GluN3A subunits as key negative regulators of GIT1 binding to mTOR. Disruption of GIT1/mTOR complexes by enhancing GluN3A expression or silencing GIT1 inhibits synaptic mTOR activation and restricts the mTOR-dependent translation of specific activity-regulated mRNAs. Conversely, GluN3A removal enables complex formation, potentiates mTOR-dependent protein synthesis, and facilitates the consolidation of associative and spatial memories in mice. The memory enhancement becomes evident with light or spaced training, can be achieved by selectively deleting GluN3A from excitatory neurons during adulthood, and does not compromise other aspects of cognition such as memory flexibility or extinction. Our findings provide mechanistic insight into synaptic translational control and reveal a potentially selective target for cognitive enhancement.

Background: UBTF is an HMGB-box DNA binding protein and a necessary Pol I/Pol II basal transcription factor. It has been found that UBTF involves in carcinogenesis and progression of a few cancers. Nevertheless, the the biological function and potential molecular mechanism of UBTF in melanoma are still not clear and need to be clarified. Methods: UBTF and GIT1 expressions in melanoma specimens and cell lines were examined by quantitative real-time PCR (qRT-PCR) and Western blot. MTT and colony formation assays were used to investigate the effects of UBTF and GIT1 on melanoma cell proliferation. Cell cycle and apoptosis assays were detected by flow cytometry. Tumor formation assay was used to analyze the effect of UBTF on melanoma growth. Bioinformatics predicting, chromatin immunoprecipitation (ChIP)-qRT-PCR and reporter gene assay were fulfilled for verifing GIT1 as UBTF targeting gene. Results: Here we reported that UBTF mRNA and protein expressions were upregulated in primary melanoma specimens and cell lines. UBTF overexpression facilitated melanoma cell proliferation and cell cycle progression and restrained. Silencing UBTF suppressed cell multiplication, cell cycle progression and tumor growth, and promoted apoptosis. UBTF expression was positively related with GIT1 expression in human melanoma tissues. It was verified that UBTF promoted GIT1 transcription in melanoma cells through binding to the promoter region of GIT1. Furthermore, GIT1 overexpression promoted melanoma cell growth and suppressed apoptosis. Knockdown of GIT1 inhibited cell multiplication and induced apoptosis. Overexpression of GIT1 eliminated the effects of silencing UBTF on melanoma cells. Importantly, UBTF activated MEK1/2-ERK1/2 signalling pathways by upregulating GIT1 expression. Conclusions: Our study demonstrates that UBTF promotes melanoma cell proliferation and cell cycle progression by promoting GIT1 transcription, thereby activating MEK1/2-ERK1/2 signalling pathways. The findings indicate that UBTF plays a crucial function in melanoma and may be a potential therapeutic target for the treatment of this disease. [ABSTRACT FROM AUTHOR]

Aim: While epithelial-mesenchymal transition (EMT) is associated with the complex morphogenetic events during embryogenesis, EMT has also been shown to play an important role in the progression of epithelial cancers. There have been few studies examining ZEB1 and SMAD protein expressions in skin tumor tissues in the literature, and there are no studies evaluating GIT1 protein expression. Materials and Methods: Thirty-seven pieces of squamous cell carcinoma(SCC), 34 pieces of basal cell carcinoma(BCC), 11 pieces of actinic keratosis(AK), 9 pieces of in situ SCC(SCCIS) and 7 pieces of normal skin tissue were included in this study. Results: A statistically significant difference was found between the SMAD1, AREB6 and GIT1 H-scores values of the individuals in the five groups. The levels of SMAD1, AREB6 and GIT1 were higher in SCC than in the control group. In binary comparisons, the SMAD1 H-score values of BCC, SCCIS groups were statistically significantly higher than normal skin. GIT1 H-score values of SCC and BCC groups were significantly higher than normal skin. Conclusion: In skin tumors, EMT is an ever-active mechanism. It is thought that this mechanism is highly controlled in SCC, a more aggressive type of cancer, than in BCC. These results suggest that the investigation of genes related to SMAD1, AREB6 and GIT1 may be useful for research into new molecular targets for the treatment and prevention of metastasis in nonmelanotic skin tumors. [ABSTRACT FROM AUTHOR]

Zitao Zhang,1 Polu Hu,2 Jin Xiong,1 Shoufeng Wang1 1Department of Orthopedics, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China; 2Nanjing Red Cross Blood Center, Nanjing 210003, China Background: GIT1, a scaffold protein with ubiquitous multi-domain, is involved in many cellular processes. In recent years, it was proved that GIT1 participated in various tumors’ growth or metastasis. However, the biological function of GIT1 in osteosarcoma is still unclear. In this study, we aimed to investigate the role and mechanism of GIT1 in osteosarcoma.Materials and methods: Human osteosarcoma tissues were obtained to investigate the distribution of GIT1. Adequate osteosarcoma cells were stably infected with lentivirus to knockdown GIT1 level and then was used to carry out cell invasion and vascular endothelial growth factor (VEGF) assay in vitro. Orthotopic femoral osteosarcoma model was constructed to investigate the growth, invasion, and angiogenesis in vivo. Western blot was used to detect extracellular signal-regulated kinase (ERK1/2) activation and hypoxia-inducible factor-1 (HIF-1α) expression.Results: In this study, we found that GIT1 was distributed in human osteosarcoma tissues and highly expressed in osteosarcoma (OS) cells. Knockdown of GIT1 inhibited cell invasion and VEGF release in vitro and suppressed tumor growth, invasion, and angiogenesis in vivo. Furthermore, knockdown of GIT1 substantially downregulated the protein levels of p-ERK and HIF-1α in OST cells and inhibition of p-ERK by PD98059 could significantly decrease the expression of HIF-1α and concentration of VEGF in GIT1-shRNA-treated cells.Conclusion: GIT1 knockdown can effectively inhibit the growth, invasion, and angiogenesis of osteosarcoma. Thus, GIT1 might act as an oncogenic factor in osteosarcoma and could be a potential molecular target for osteosarcoma gene therapy. Keywords: GIT1, osteosarcoma, angiogenesis, orthotopic femoral osteosarcoma model, ERK1/2, HIF-1α

Background/Aims: Periodic mechanical stress has been shown to promote extracellular matrix (ECM) synthesis and cell migration of nucleus pulposus (NP) cells, however, the mechanisms need to be fully elucidated. The present study aimed to investigate the signal transduction pathway in the regulation of NP cells under periodic mechanical stress. Methods: Primary rat NP cells were isolated and seeded on glass slides, and then treated in our self-developed periodic stress field culture system. To further explore the mechanisms, data were analyzed by scratch-healing assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, and co-immunoprecipitation assay. Results: Under periodic mechanical stress, the mRNA expression of ECM collagen 2A1 (Col2A1) and aggrecan, and migration of NP cells were significantly increased (P < 0.05 for each), associating with increases in the phosphorylation of Src, GIT1, and ERK1/2 (P < 0.05 for each). Pretreatment with the Src inhibitor PP2 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each), while the phosphorylation of GIT1 and ERK1/2 were inhibited. ECM synthesis, cell migration, and phosphorylation of ERK1/2 were inhibited after pretreatment with the small interfering RNA for GIT1 in NP cells under periodic mechanical stress (P < 0.05 for each), whereas the phosphorylation of Src was not affected. Pretreatment with the ERK1/2 inhibitor PD98059 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each). Co-immunoprecipitation assay showed that there was a direct interaction between Src and GIT1 and between GIT1 and ERK1/2. Conclusion: In conclusion, periodic mechanical stress induced ECM expression and migration of NP cells via Src-GIT1-ERK1/2 signaling pathway, playing an important role in regulation of NP cells.

Background/Aims: The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin β1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin β1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin β1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin β1-FAK(Tyr397)-ERK1/2.

• Establish animal model of METH-induced behavioural sensitization. • Screen DEGs by RNA-seq, and carry out GO and pathway analysis for DEGs. • Choose Fas and GIT1 as new targets of METH-induced behavioural sensitization. • Verify Fas and GIT1 affected behavioural sensitization to METH via the MEK1-Erk1/2-CREB pathway. Repeated methamphetamine (METH) administration in mice readily produces behavioural sensitization, but the underlying mechanisms remain elusive. The present research aimed to identify new targets affecting METH-induced behavioural sensitization. We first established a mouse model of METH-induced behavioural sensitization. To characterize the animal model, we performed behavioural tests at different stages of behavioural sensitization and simultaneously detected changes in several neurotransmitters in the prefrontal cortex (PFC). Next, we perfromed RNA sequencing (RNA-seq) to screen new targets, which were subsequently and verified by RT-PCR and western blot. Finally, we confirmed the roles of the new targets in METH-induced behavioural sensitization by injection of overexpressed lentiviruses and further detected related protein levels by western blot and histological changes by haematoxylin and eosin (HE) staining. We successfully established a mouse model of METH-induced behavioural sensitization. The locomotor activities of the mice changed at different stages of sensitization, accompanied by changes in the levels of DA, 5-HT, GABA and glutamate. For RNA-seq analysis, we chose Fas as target, meanwhile, we chose GIT1 as target through literature. The detection of gene expression by RT-PCR indicated that METH-sensitized mice exhibited decreased levels of Fas, MEK1 and CREB and increased levels of Erk1/2 in the PFC. Western blot analysis revealed decreased Fas, GIT1, MEK1 and phosphorylated CREB levels and increased phosphorylated Erk1/2 levels in METH-sensitized mice. Injection of Fas and GIT1 injection showed that overexpression of Fas and GIT1 inhibited the induction of METH sensitization and reversed the changes in neurotransmitter levels and related protein levels, including MEK1, phosphorylated CREB and phosphorylated Erk1/2, in METH-sensitized mice. Overexpression of Fas and GIT1 also reduced histological lesions induced by METH. The findings indicated that the development of behavioural sensitization to METH may be mediated by Fas and GIT1 through the MEK1-Erk1/2-CREB pathway. [ABSTRACT FROM AUTHOR]

Abstract Background Although tibial plateau fracture is an uncommon injury, its regulation is challenging and there are some influencing factors, including the effects of severe bone displacement, depression and cancellous bone cartilage, and inevitable cartilage damage. And GIT1 plays an important role in bone mass and 78 osteoblast cell migration. Methods The study used 72 C57/BL6 mice. A tibial plateau fracture model was established by using mice with the same number of GIT1 gene deletions (the experimental group) and their wild-type littermates (the control group). Joint and bone callus recovery were evaluated by X-ray and CT thin layer scans. Micro CT assay and histomorphometry were conducted in order to evaluate the volume of newly formed blood vessels. Type II collagen expression in tibial tissues after tibial plateau fracture were detected by immunohistochemistry after 7, 14 and 21 days. The number of proliferating cell nuclear antigen (PCNA) positive cells after tibial plateau fracture was tested by immunohistochemistry after 14 and 21 days. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was conducted after 14 and 21 days in order to test chondrocyte apoptosis in tibial tissues after tibial plateau fracture. Results The GIT1 gene deletion group mice spent less time on the rotating rod than the control group mice (P

In spinal cord ischemia‐reperfusion (I/R) injury, large amounts of reactive oxygen species can cause mitochondrial damage. Therefore, mitophagy acts as the main mechanism for removing damaged mitochondria and protects nerve cells. This study aimed to illustrate the important role of GPCR kinase 2–interacting protein‐1 (GIT1) in mitophagy in vivo and in vitro. The level of mitophagy in the neurons of Git1 knockout mice was significantly reduced after ischemia‐reperfusion. However, the overexpression of adeno‐associated virus with Git1 promoted mitophagy and inhibited the apoptosis of neurons. GIT1 regulated the phosphorylation of Beclin‐1 in Thr119, which could promote the translocation of Parkin to the mitochondrial outer membrane. This process was independent of PTEN‐induced kinase 1 (PINK1), but it could not rescue the role in the absence of PINK1. Overall, GIT1 enhanced mitophagy and protected neurons against ischemia‐reperfusion injury and, hence, might serve as a new research site for the protection of ischemia‐reperfusion injury. [ABSTRACT FROM AUTHOR]

Abstract G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) is a scaffold protein that plays a vital role in bone modeling and remodeling during osteogenesis coupled with angiogenesis. Recent studies have shown that a specialized subset of vascular endothelium strongly positive for CD31 and Endomucin (CD31hiEmcnhi) is coupled with anabolic bone formation. Based on our previous finding that GIT1 knockout (GIT1 KO) mice have impaired angiogenesis and bone formation, we hypothesized that GIT1 affects formation of the CD31hiEmcnhi vessel subtype. In the current study, GIT1 knockout (GIT1 KO) mice displayed a significant decrease in trabecular bone mass and CD31hiEmcnhi vessel number, compared to their wild-type counterparts. In the fracture healing mouse model, GIT1 KO mice contained a lower number of CD31hiEmcnhi vessels in fracture callus at days 7 and 14. However, no significant differences in the number of preosteoclasts in bone marrow, trabecular bone and callus in GIT1 KO mice were observed, compared with wild-type mice. Notably, concentrations of serum platelet-derived growth factor-BB(PDGF-BB) secreted by preosteoclasts associated with CD31hiEmcnhi vessel formation were lower in GIT1 KO mice. In addition, PDGF-BB-associated expression of phosphorylated extracellular signal-regulated kinase- 1/2 (ERK1/2) and specificity protein 1 (SP1) was significantly decreased in preosteoclasts of GIT1 KO mice. These results collectively suggest that GIT1 is a critical participant in formation of the CD31hiEmcnhi vessel subtype, highlighting a novel biologic function of this scaffold protein in preosteoclasts. [ABSTRACT FROM AUTHOR]

GIT1, a G-protein-coupled receptor kinase interacting protein, has been reported to be involved in neurite outgrowth. However, the neurobiological functions of the protein remain unclear. In this study, we found that GIT1 was highly expressed in the nervous system, and its expression was maintained throughout all stages of neuritogenesis in the brain. In primary cultured mouse hippocampal neurons from GIT1 knockout mice, there was a significant reduction in total neurite length per neuron, as well as in the average length of axon-like structures, which could not be prevented by nerve growth factor treatment. Overexpression of GIT1 significantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice. The GIT1 N terminal region, including the ADP ribosylation factor-GTPase activating protein domain, the ankyrin domains and the Spa2 homology domain, were sufficient to enhance axonal extension. Importantly, GIT1 bound to many tubulin proteins and microtubule-associated proteins, and it accelerated microtubule assembly in vitro. Collectively, our findings suggest that GIT1 promotes neurite outgrowth, at least partially by stimulating microtubule assembly. This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neurological diseases.

Background: The outcome for oestrogen receptor positive (ER+) breast cancer patients has improved greatly in recent years largely due to targeted therapy. However, the presence of involved multiple synchronous lymph nodes remains associated with a poor outcome. Consequently, these patients would benefit from the identification of new prognostic biomarkers and therapeutic targets. The expression of G-protein-coupled receptor kinase-interacting protein 1 (GIT1) has recently been shown to be an indicator of advanced stage breast cancer. Therefore, we investigated its expression and prognostic value of GIT1 in a cohort of 140 ER+ breast cancer with synchronous lymph node involvement. Methods: Immunohistochemistry was employed to assess GIT1 expression in a tissue microarray (TMA) containing duplicate non-adjacent cores with matched primary tumour and lymph node tissue (n=140). GIT1 expression in tumour cells was scored and statistical correlation analyses were carried out. Results: The results revealed a sub-group of patients that displayed discordant expression of GIT1 between the primary tumour and the lymph nodes (i.e. spatial intratumoural heterogeneity). We observed that loss of GIT1 expression in the tumour cells of the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time. Moreover, multivariate analysis demonstrated that GIT1 expression was an independent prognostic indicator. Conclusions: GIT1 expression enabled the identification of a sub-class of ER+ patients with lymph node metastasis that have a particularly poor prognostic outcome. We propose that this biomarker could be used to further stratify ER+ breast cancer patients with synchronous lymph node involvement and therefore facilitate adjuvant therapy decision making.

Background: The outcome for oestrogen receptor positive (ER+) breast cancer patients has improved greatly in recent years largely due to targeted therapy. However, the presence of involved multiple synchronous lymph nodes remains associated with a poor outcome. Consequently, these patients would benefit from the identification of new prognostic biomarkers and therapeutic targets. The expression of G-protein-coupled receptor kinase-interacting protein 1 (GIT1) has recently been shown to be an indicator of advanced stage breast cancer. Therefore, we investigated its expression and prognostic value of GIT1 in a cohort of 140 ER+ breast cancer with synchronous lymph node involvement. Methods: Immunohistochemistry was employed to assess GIT1 expression in a tissue microarray (TMA) containing duplicate non-adjacent cores with matched primary tumour and lymph node tissue (n=140). GIT1 expression in tumour cells was scored and statistical correlation analyses were carried out. Results: The results revealed a sub-group of patients that displayed discordant expression of GIT1 between the primary tumour and the lymph nodes (i.e. spatial intratumoural heterogeneity). We observed that loss of GIT1 expression in the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time. Moreover, multivariate analysis demonstrated that GIT1 expression was an independent prognostic indicator. Conclusions: GIT1 expression enabled the identification of a sub-class of ER+ patients with lymph node metastasis that have a particularly poor prognostic outcome. We propose that this biomarker could be used to further stratify ER+ breast cancer patients with synchronous lymph node involvement and therefore facilitate adjuvant therapy decision making.

Previous studies indicate that miR-149 could both inhibit and promote the development of human cancer depending on the tumor type. GIT1 was found to play an important role in regulating cell migration. However, the specific function of miR-149-5p and GIT1 in the progression of medullary thyroid carcinoma (MTC) remains unknown. The purpose of this study was to confirm the function of miR-149-5p in MTC and explore its downstream regulation. Moreover, miR-149-5p level in MTC was detected via RT-quantitative PCR (RT-qPCR). GIT1 expression levels were assessed by RT-qPCR and western blot analysis. The cell proliferation and invasion were detected through MTT or Transwell assay respectively. In addition, miR-149-5p was identified to directly target GIT1 in MTC via dual luciferase assay. The results suggested that miR-149-5p level was obviously declined in MTC. Functionally, miR-149-5p overexpression inhibited proliferation and invasion. Moreover, miR-149-5p directly targeted GIT1 and was negatively associated with its expression in MTC. Conversely, GIT1 expression was obviously increased in MTC. GIT1 overexpression partially reversed the inhibitory action of miR-149-5p in MTC. miR-149-5p suppressed the proliferation and invasion of MTC cells through targeting GIT1, which would create new therapeutic avenues for MTC treatment. [ABSTRACT FROM AUTHOR]

Potent androgen receptor pathway inhibition (ARPI) therapies have given rise to a lethal, aggressive subtype of castration‐resistant prostate cancer (CRPC) called treatment‐induced neuroendocrine prostate cancer (t‐NEPC). Now, t‐NEPC poses a major clinical problem as approximately 20% of CRPC cases bear this subtype—a rate of occurrence that is predicted to rise with the widespread use of ARPI therapies. Unfortunately, there are no targeted therapies currently available to treat t‐NEPC as the origin and molecular underpinnings of t‐NEPC development remain unclear. In the present study, we identify that RNA splicing of the G protein‐coupled receptor kinase‐interacting protein 1 (GIT1) gene is a unique event in t‐NEPC patients. Specifically, upregulation of the GIT1‐A splice variant and downregulation of the GIT1‐C variant expressions are associated with t‐NEPC patient tumors, patient‐derived xenografts, and cell models. RNA‐binding assays show that RNA splicing of GIT1 is directly driven by SRRM4 and is associated with the neuroendocrine phenotype in CRPC cohorts. We show that GIT1‐A and GIT1‐C regulate differential transcriptomes in prostate cancer cells, where GIT1‐A regulates genes associated with morphogenesis, neural function, environmental sensing via cell‐adhesion processes, and epigenetic regulation. Consistent with our transcriptomic analyses, we report opposing functions of GIT1‐A and GIT1‐C in the stability of focal adhesions, whereby GIT1‐A promotes focal adhesion stability. In summary, our study is the first to report that alternative RNA splicing of the GIT1 gene is associated with t‐NEPC and reprograms its function involving FA‐mediated signaling and cell processes, which may contribute to t‐NEPC development. We report that the alternative RNA splicing of the GIT1 gene is associated with t‐NEPC progression using patient tumor samples, PDX models, and cell models. We demonstrate that GIT1‐A expression is upregulated in NEPC models, whereas expression of GIT1‐C is downregulated when compared to AdPC models, a mechanism regulated by SRRM4‐mediated RNA alternative splicing. Comprehensive transcriptomic data reveal distinct molecular changes in GIT1‐A and GIT1‐C, whereby bioinformatic analyses predict that the GIT1‐A transcriptome is enriched with gene sets implicated in t‐NEPC progression, such as morphogenesis, neural function, environmental sensing via cell‐adhesion processes, and epigenetic regulation. Consistent with our transcriptomic analyses, we report opposing functions of GIT1‐A and GIT1‐C in the stability of focal adhesions, whereby GIT1‐A promotes focal adhesion stability. [ABSTRACT FROM AUTHOR]

GPCR kinase 2-interacting protein-1 (GIT1) is a scaffold protein that plays an important role in cell adaptation, proliferation, migration, and differentiation; however, the role of GIT1 in the regulation of neuronal death after spinal cord injury remains obscure. Here, we demonstrate that GIT1 deficiency remarkably increased neuronal apoptosis and enhanced JNK/p38 signaling, which resulted in stronger motor deficits by ischemia-reperfusion in vivo, consistent with the finding of oxygen-glucose deprivation/reoxygenation-induced neuronal injury in vitro. After treatment with JNK and p38 inhibitors, abnormally necroptotic cell death caused by GIT1 knockdown could be partially rescued, with the recovery of neuronal viability, which was still poorer than that in control neurons. Meanwhile, overactivation of JNK/p38 after GIT1 depletion was concomitant with excessive activity of apoptosis signal-regulating kinase-1 (ASK1) that could be abolished by ASK1 silencing in HEK293T cells. Finally, GIT1 could disrupt the oligomerization of ASK1 via interaction between the synaptic localization domain that contains the coiled-coil (CC)-2 domain of GIT1 and the C-terminal CC domain of ASK1. It suppressed the autophosphorylation of ASK1 and led to decreasing activity of the ASK1/JNK/p38 pathway. These data reveal a protective role for GIT1 in neuronal damage by modulating ASK1/JNK/p38 signaling. [ABSTRACT FROM AUTHOR]

Background/Aims: Periodic mechanical stress has been shown to promote extracellular matrix (ECM) synthesis and cell migration of nucleus pulposus (NP) cells, however, the mechanisms need to be fully elucidated. The present study aimed to investigate the signal transduction pathway in the regulation of NP cells under periodic mechanical stress. Methods: Primary rat NP cells were isolated and seeded on glass slides, and then treated in our self-developed periodic stress field culture system. To further explore the mechanisms, data were analyzed by scratch-healing assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, and co-immunoprecipitation assay. Results: Under periodic mechanical stress, the mRNA expression of ECM collagen 2A1 (Col2A1) and aggrecan, and migration of NP cells were significantly increased (P < 0.05 for each), associating with increases in the phosphorylation of Src, GIT1, and ERK1/2 (P < 0.05 for each). Pretreatment with the Src inhibitor PP2 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each), while the phosphorylation of GIT1 and ERK1/2 were inhibited. ECM synthesis, cell migration, and phosphorylation of ERK1/2 were inhibited after pretreatment with the small interfering RNA for GIT1 in NP cells under periodic mechanical stress (P < 0.05 for each), whereas the phosphorylation of Src was not affected. Pretreatment with the ERK1/2 inhibitor PD98059 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each). Co-immunoprecipitation assay showed that there was a direct interaction between Src and GIT1 and between GIT1 and ERK1/2. Conclusion: In conclusion, periodic mechanical stress induced ECM expression and migration of NP cells via Src-GIT1-ERK1/2 signaling pathway, playing an important role in regulation of NP cells. [ABSTRACT FROM AUTHOR]

Background: Exosomes secreted by human mesenchymal stem cells (hMSCs) have been shown to promote cartilage regeneration. This study aimed to explore whether exosomal lncRNA-KLF3-AS1 derived from hMSCs can promote chondrocyte proliferation via miR-206/GIT1 axis in osteoarthritis (OA). Methods: hMSCs and MSC-derived exosomes (MSC-exo) were prepared for morphological observation and identification by transmission electron microscopy (TEM) and flow cytometry. IL-1β-induced OA chondrocytes and collagenase-induced mouse OA model were established for the further experiments. Luciferase activity assay was performed to test whether miR-206 could bind to KLF3-AS1 or GIT1. Cell proliferation and apoptosis were evaluated by CCK-8 assay and flow cytometry, respectively. Results: MSC-Exos increased chondrogenic genes Col2a1 (type II collagen alpha 1) and aggrecan, decreased hondrocyte hypertrophy markers MMP-13 (matrix metalloproteinase-13) and Runx2 (runt-related transcription factor 2) in chondrocytes isolated from OA model mice. Furthermore, MSC-Exos attenuated IL-1β-induced chondrocyte proliferation inhibition and apoptosis induction. Moreover, MSCKLF3-AS1-Exos (exosomes derived from KLF3-AS1-overexpressing-MSCs) ameliorated IL-1β-induced chondrocyte injury. Results also demonstrated that KLF3-AS1 acted as a competitive endogenous RNA (ceRNA) by sponging miR-206 to facilitate GIT1 expression. In addition, miR-206 overexpression and GIT1 knockdown reversed MSCKLF3-AS1-Exos-mediated attenuation of chondrocyte injury. Conclusion: Exosomal KLF3-AS1 derived from MSCs involved in MSC-Exos-mediated chondrocyte proliferation induction and chondrocyte apoptosis inhibition via miR-206/GIT1 axis. Abbreviation: G-protein-coupled receptor kinase interacting protein-1 (GIT1) [ABSTRACT FROM AUTHOR]

Background/Aims: The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin β1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin β1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin β1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin β1-FAK(Tyr397)-ERK1/2. [ABSTRACT FROM AUTHOR]

MiRNAs are emerging as critical regulators in carcinogenesis and tumor progression. Recently, miR-149 has been demonstrated as a tumor suppressor in several cancers, but its functions in the context of cervical cancer remains unknown. The objective of this study is to investigate the clinicopathological and prognostic values of miR-149 expression and its roles in cervical cancer progression. In this study, miR-149 expression is decreased in CC tissues and cell lines compare with paired normal tissues and normal epithelial cell, respectively. Moreover, the restoration of miR-149 expression inhibited cell proliferation, migration, invasion and promoted cell apoptosis in vitro. We further proved that miR-149 overexpression suppresse the growth of cervical cancer cells in vivo using a mouse xenograft model. Dual luciferase assays identified the GIT1 as a novel direct target of miR-149. To identify the mechanisms, we investigated the PI3K/AKT/mTOR pathway and found that the expression of PI3K, AKT and mTOR were suppressed in cells which were transfected with miR-149 mimics. Taken together, these results indicate that miR-149 is involved in the carcinogenesis and development of cervical cancer by targeting GIT1 via PI3K/AKT/mTOR pathway. Given these, our data suggest that miR-149 functions as a tumor suppressor and may serve as a biomarker or useful therapeutic target of cervical cancer. [ABSTRACT FROM AUTHOR]

Breast cancer remains a major cause of cancer-related death in women worldwide. Dysregulation of microRNAs (miRNAs) is involved in the initiation and progression of breast cancer. Moreover, it was found that GIT1 was widely involved in the development of many human cancers. Herein, we aimed to investigate the expression changes of miR-149* and GIT1 and the functional effects of miR-149*/GIT1 link in breast cancer. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot (WB) were used to examine the expression levels of miR-149* and GIT1. Dual luciferase reporter assay was utilized to confirm the target interaction between miR-149* and GIT1. The biological functions, including cell proliferation, invasion, and migration, of miR-149* and GIT1 were determined by MTT assay and Transwell assays, respectively. Eventually, the tumor xenograft model in nude mice injected with stable transfected MDA-MB-231 cells was established to verify the effects of miR-149* and GIT1 on tumor growth. Our results showed that miR-149* expression was decreased, whereas GIT1 expression was increased in clinical samples of breast cancer. Based on the inverse expression trend between miR-149* and GIT1, we further demonstrated that miR-149* indeed directly targets GIT1. Subsequently, it was observed that inhibition of miR-149* significantly promoted cell proliferation, invasion, and migration, but the ability of cell proliferation, invasion, and migration was obviously declined after silencing of GIT1 in MDA-MB-231 cells transfected with miR-149* mimic and/or si-GIT1. Finally, it was also found that elevated miR-149* decelerated the tumor growth, while restored GIT1 accelerated the tumor growth in nude mice after 35 days of tumor xenograft. Collectively, these findings concluded that miR-149* might exert a tumor suppressive role in breast cancer by targeting GIT1. [ABSTRACT FROM AUTHOR]

Background: Despite G-protein-coupled receptor kinase-interacting protein-1 (GIT1) being recognized as a new promoter gene in some types of cancer, its effect on human pan-cancers and liver hepatocellular carcinoma (LIHC) remains unclear., Objectives: To elucidate the molecular mechanisms of GIT1 in pan-cancer and LIHC., Material and Methods: Various bioinformatics approaches were utilized to elucidate the oncogenic effects of GIT1 on human pan-cancers., Results: The GIT1 was aberrantly expressed in pan-cancers and associated with the clinical stage. Moreover, the upregulation of GIT1 expression was indicative of poor overall survival (OS) in patients with LIHC, skin cutaneous melanoma (SKCM) and uterine corpus endometrial carcinoma (UCEC), as well as of poor disease-free survival (DFS) in patients with LIHC and UCEC. Furthermore, GIT1 levels were correlated with cancer-associated fibroblasts (CAFs) in adrenocortical carcinoma (ACC), cervical squamous cell carcinoma (CESC) and LIHC. The analysis of single-cell sequencing data revealed an association of GIT1 levels with apoptosis, cell cycle and DNA damage. In addition, multivariate Cox analysis indicated that high GIT1 levels were an independent risk factor for shorter OS in patients with LIHC. Finally, the gene set enrichment analysis revealed INFLAMMATORY&#95;RESPONSE pathway and IL2&#95;STAT5&#95;SIGNALING to be the most enriched in LIHC., Conclusions: Our data demonstrate the oncogenic effects of GIT1 on various cancers. We believe that GIT1 can serve as a biomarker for LIHC.

As a critical regulator of cell growth, the mechanistic target of rapamycin (mTOR) protein operates as part of two molecularly and functionally distinct complexes. Here in, we demonstrate that mTOR complex molecular composition varies in different somatic tissues. In astrocytes and neural stem cells, we identified G-protein-coupled receptor kinase-interacting protein 1 (GIT1) as a novel mTOR-binding protein, creating a unique mTOR complex lacking Raptor and Rictor. Moreover, GIT1 binding to mTOR is regulated by AKT activation and is essential for m TOR- mediated astrocyte survival. Together, these data reveal that mTOR complex function is partly dictated by its molecuflar composition in different cell types. [ABSTRACT FROM AUTHOR]

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, a-PIX and β-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT-PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT-PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT- PIX complex in health and disease. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD), known as a neurodegenerative disease, is characterized by movement disorders, with increasing age being the predominant risk factor for its development. Mangiferin, a bioactive compound isolated from mango, shows potent neuroprotection. In our work, we investigated the neuroprotection and mechanisms of mangiferin against PD. We established PD models by treating SH-SY5Y cells with rotenone and mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and investigated the therapeutic effects of mangiferin. Our results showed that mangiferin exhibited a cell-protective effect. Mangiferin also improved the motor behavior and attenuated the activation of microglia and astrocytes in MPTP mice. In addition, mangiferin decreased reactive oxygen species (ROS) levels and increased glutathione (GSH) and superoxide dismutase (SOD). Mangiferin also markedly activated GIT1, p-ERK, Nrf2, HO-1, and SOD expression and inhibited Keap1 expression in vitro and in vivo . To further investigate the role of GIT1, GIT1 siRNA was used. In the presence of GIT1 siRNA, the neuroprotection of mangiferin in PD was weakened. Our results indicate that mangiferin exhibited its therapeutic effect against PD by regulating GIT1 and its downstream Keap1/Nrf2 pathways. Our studies exhibited that mangiferin showed neuroprotection in PD, and its main target was GIT1. What is more, mangiferin could reduce the oxidative stress of PD by targeting GIT1 and its downstream Keap1/Nrf2 pathways. These indicated that mangiferin is a good candidate for PD therapy. However, the role of p-ERK in mangiferin-treated PD requires further investigation.

Integrins play an important role in cancer growth and metastasis. This study aimed at determining the predictive ability of integrins and associated genes identified through molecular network in clear cell renal cell carcinoma. A total of 525 patients with ccRCC from The Cancer Genome Atlas (TCGA) cohorts were collected in this study. The expression profile of integrins and related genes were obtained from the TCGA RNAseq database. Clinicopathological characteristics, including age, gender, tumor size, tumor node metastasis (TNM), tumor grade, stage, laterality, and overall survival were collected. Cox proportional hazards regression model as well as Kaplan-Meier curve were used to assess the relative factors. Genes of integrin family that showed certain correlations with overall survival (OS) were further validated in the Fudan University Shanghai Cancer Center (FUSCC) cohort. In the TCGA cohort, after Cox proportional hazards analysis, ITGA2B (hazards ratio (HR) = 1.232, 95 % CI 1.097 to 1.383) and ITGA8 (HR = 0.804, 95 % CI 0.696 to 0.930) were shown predictive of ccRCC prognosis. Low ITGA8 expression was associated with poor prognosis for OS (log-rank test, p < 0.0001), while high level of ITGA2B expression was correlated with poor prognosis for OS (log-rank test, p < 0.0001). This finding was validated in FUSCC cohort (log-rank test, all p < 0.05). As a result, low ITGA8 expression was associated with poor prognosis for OS (log-rank test, p = 0.0053), while high level of ITGA2B expression was correlated with poor prognosis for OS (log-rank test, p < 0.0001). Plus, low ITGA8 expression was associated with poor prognosis for disease-free survival (DFS) in the TCGA cohort (log-rank test, p < 0.0001). In the gene cluster network analysis, GIT1 and SHC1 associated with ITGA2B and ITGA8 were identified as independent predictive factors of overall survival of ccRCC. ITGA2B, ITGA8, GIT1, and SHC1 were identified as independent prognostic factors of overall survival of ccRCC. This method may act as a tool to reveal more prognostic-associated genes in ccRCC. [ABSTRACT FROM AUTHOR]

Aim: Multiple studies have revealed that G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is overexpressed in many cancers and facilitates tumor progression. However, the role of GIT1 in hepatocellular carcinoma (HCC) remains unclear. Methods: GIT1 expression was detected in cell lines and 130 pairs of HCC and matched adjacent noncancerous samples. Transwell assay, flow cytometry, caspase 3/7 activity assay, 5-bromodeoxyuridine cell proliferation assay, and 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay were used to assess invasion, migration, apoptosis, and proliferation of HCC cells. Furthermore, GIT1 expression was detected by immunohistochemistry to evaluate its correlation with phospho-extracellular signal-regulated kinase (p-ERK)1/2. The regulatory effect of GIT1 on ERK1/2, p-ERK1/2, and matrix metalloproteinase-9 (MMP9) in HCC cells was confirmed by immunoblotting. Results: In this study, we demonstrated that GIT1 was more highly expressed in HCC samples than that in non-HCC samples, and overexpression of GIT1 was correlated with clinicopathological features of poor prognosis. Clinical analysis demonstrated that GIT1 is an independent prognostic biomarker for predicting overall survival and disease-free survival of patients with HCC. In vitro studies showed that downregulation of GIT1 facilitated HCC cell apoptosis and repressed HCC cell invasion, migration, and proliferation. Overexpression of GIT1 is associated with p-ERK1/2 amplification in HCC tissues. Moreover, downregulation of GIT1 resulted in inactivation of ERK signaling and downregulation of MMP9. Conclusion: Our findings indicate that GIT1 is an independent prognostic biomarker and facilitates HCC progression via activating ERK/MMP9 signaling. [ABSTRACT FROM AUTHOR]

Non-small-cell lung cancer (NSCLC) is one of the most common and lethal malignant tumours worldwide with a poor 5-year survival rate. Recent studies indicated that miRNAs have been involved in the tumorigenic driver pathways in NSCLC, but the relevant molecular mechanisms are not well-understood. In this study, we investigated the biological functions and molecular mechanisms of miR-138 in human NSCLC. The effects of miR-138 on the NSCLC cell growth and epithelial-mesenchymal transition (EMT) were first examined. Then the targeting connections of miR-138 with G-protein-coupled receptor kinase-interacting protein 1 (GIT1) and semaphorin 4C (SEMA4C) were confirmed by dual luciferase reporter assays. Finally, the effects of GIT1 and SEMA4C on the NSCLC cell growth and EMT were investigated respectively. We found that the ectopic expression of miR-138 resulted in a significant inhibition of NSCLC growth and reversion of EMT. GIT1 and SEMA4C were identified as two novel targets of miR-138. Furthermore, GIT1 and SEMA4C knockdown inhibited the cell growth and reversed EMT, just like the effects of miR-138 overexpression on NSCLC cells, whereas ectopic expression of GIT1 and SEMA4C partly rescued the suppressive effects of miR-138 in NSCLC cells. These data represent a crucial step towards the understanding of the novel roles and molecular mechanism of miR-138, GIT1 and SEMA4C in NSCLC progression, which may provide some new targets or prognostic biomarkers for NSCLC treatment, thus having implications in translational oncology. [ABSTRACT FROM AUTHOR]

Endochondral ossification, an important stage of fracture healing, is regulated by a variety of signaling pathways. Transforming growth factor β (TGFβ) superfamily plays important roles and comprises TGFβs, bone morphogenetic proteins (BMPs), and growth differentiation factors. TGFβs primarily regulate cartilage formation and endochondral ossification. BMP2 shows diverse efficacy, from the formation of skeleton and extraskeletal organs to the osteogenesis and remodeling of bone. G-protein-coupled receptor kinase 2-interacting protein-1 (GIT1), a shuttle protein in osteoblasts, facilitates fracture healing by promoting bone formation and increasing the secretion of vascular endothelial growth factor. Our study examined whether GIT1 regulates fracture healing through the BMP2 signaling pathway and/or through the TGFβ signaling pathway. GIT1 knockout (KO) mice exhibited delayed fracture healing, chondrocyte accumulation in the fracture area, and reduced staining intensity of phosphorylated Smad1/5/8 (pSmad1/5/8) and Runx2. Endochondral mineralization diminished while the staining intensity of phosphorylated Smad2/3 (pSmad2/3) showed no significant change. Bone marrow mesenchymal stem cells extracted from GIT1 KO mice showed a decline of pSmad1/5/8 levels and of pSmad1/5/8 translocated into the cell nucleus after BMP2 stimulus. We detected no significant change in the pSmad2/3 level after TGFβ1 stimulus. Data obtained from reporter gene analysis of C3H10T1/2 cells cultured in vitro confirmed these findings. GIT1-siRNA inhibited transcription in the cell nucleus via pSmad1/5/8 after BMP2 stimulus but had no significant effect on transcription via pSmad2/3 after TGFβ1 stimulus. Our results indicate that GIT1 regulates Smad1/5/8 phosphorylation and mediates BMP2 regulation of Runx2 expression, thus affecting endochondral ossification at the fracture site. [ABSTRACT FROM AUTHOR]

The PAK2/βPIX/GIT1 (p21-activated kinase 2/PAK-interacting exchange factor-β/G protein-coupled receptor kinase-interactor 1) complex has been shown to distribute to both membrane ruffles and focal adhesions of cells, where it plays an important role in regulating focal adhesion turnover. However, the detailed mechanism underlying this regulation is largely unknown. We previously reported that MYO18Aα interacts via its carboxyl terminus with the PAK2/βPIX/GIT1 complex through direct binding to βPIX, and that knockdown of MYO18Aα in epithelial cells causes accumulation of the complex in focal adhesions and decreased cell migration ability (Hsu et al., 2010). The current study characterized the detailed MYO18Aα–βPIX interaction mechanism and the biological significance of this interaction. We found that deletion of the carboxyl-terminal globular domain of MYO18Aα profoundly altered the cellular localization of βPIX and inhibited cell migration. βPIX interacts through its most carboxyl-terminus, PAWDETNL (639–646), with MYO18Aα and partially colocalized with MYO18Aα in membrane ruffles of cells, whereas βPIX 1–638 , a mutant with deletion of PAWDETNL, accumulated in focal adhesions. Both focal adhesion numbers and area in βPIX 1–638 -expressing cells were greater than those in cells expressing wild-type βPIX FL . Further experiments using deletion mutants of MYO18A and βPIX showed that disruption of MYO18A–βPIX interaction not only impaired cell motility but also decreased Rac1 activity. Collectively, our data unravel the interaction regions between MYO18A and βPIX and provide evidence for the critical role of this interaction in regulating cellular localization of βPIX, Rac1 activity, and adhesion and migration in epithelial cells. [ABSTRACT FROM AUTHOR]

There are many growth factors contributing to fracture healing after bone fractures. Platelet-derived growth factor (PDGF) released from platelets is a factor promoting cell division and proliferation, and first appears around the sites of fractures. Culture of chondrocytes in vitro are stimulated by PDGF to proliferation, its presence being upregulated in the extracellular matrix of cartilage; the main components include aggrecan and type II collagen. PDGF induces the expression of G the protein-coupled receptor kinase interacting protein 1 (GIT1), promoting Rac1 and ERK1/2 phosphorylation. Both knocking down GIT1 expression by siRNA and blocking phosphorylation of Rac1 inhibit this induced proliferation of chondrocyte. GIT1 and Rac1 control each other, having a synergistic effect on activation of the ERK1/2 pathway. The results suggest that PDGF regulates chondrocyte proliferation through activation of ERK1/2 pathway by upregulation of GIT1 expression and Rac1 phosphorylation. [ABSTRACT FROM AUTHOR]