Klf2-Vav1-Rac1 axis promotes axon regeneration after peripheral nerve injury.

Increasing the intrinsic regeneration potential of neurons is the key to promote axon regeneration and repair of nerve injury. Therefore, identifying the molecular switches that respond to nerve injury may play critical role in improving intrinsic regeneration ability. The mechanisms by which injury unlocks the intrinsic axonal growth competence of mature neurons are not well understood. The present study identified the key regulatory genes after sciatic nerve crush injury by RNA sequencing (RNA-Seq) and found that the hub gene Vav1 was highly expressed at both early response and regenerative stages of sciatic nerve injury. Furthermore, Vav1 was required for axon regeneration of dorsal root ganglia (DRG) neurons and functional recovery. Krüppel-like factor 2 (Klf2) was induced by retrograde Ca2+ signaling from injured axons and could directly promote Vav1 transcription in adult DRG neurons. The increased Vav1 then promoted axon regeneration by activating Rac1 GTPase independent of its tyrosine phosphorylation. Collectively, these findings break through previous limited cognition of Vav1, and first reveal a crucial role of Vav1 as a molecular switch in response to axonal injury for promoting axon regeneration, which might further serve as a novel molecular therapeutic target for clinical nerve injury repair.

KDM6A-ARHGDIB axis blocks metastasis of bladder cancer by inhibiting Rac1.

BACKGROUND: KDM6A, a histone demethylase, is frequently mutated in bladder cancer (BCa). However, the role and detailed molecular mechanism of KDM6A involved in bladder cancer progression remains unknown. METHODS: Tissue specimens were used to determine the expression levels and prognostic values of KDM6A and ARHGDIB. The MTT, colony formation, wound healing and Transwell migration and invasion assays were employed to detect the BCa cell proliferation, migration and invasion, respectively. Chemotaxis of macrophages was used to evaluate the ability of KDM6A to recruit macrophages. A subcutaneous tumour model and tail vein tumour injection in nude mice were used to assess the role of KDM6A in vivo. RNA sequencing, qPCR, Western blot, ChIP and phalloidin staining assay were performed to investigate the molecular functions of KDM6A. Dual-luciferase reporter assay was used to determine the effects of KDM6A and FOXA1 on the promoters of the ARHGDIB and KDM6A. RESULTS: We showed that the KDM6A inhibited the motility and invasiveness of the BCa cells. Mechanistically, KDM6A promotes the transcription of ARHGDIB by demethylating histone H3 lysine di/trimethylation (H3K27me2/3) and consequently leads to inhibition of Rac1. EZH2, which catalyses the methylation of H3K27, functions to silence ARHGDIB expression, and an EZH2 inhibitor can neutralize the metastatic effect caused by KDM6A deficiency. Furthermore, we demonstrated that FOXA1 directly binds to the KDM6A promoter and thus transactivates KDM6A, leading to diminished metastatic potential. CONCLUSION: Our findings establish the critical role of the FOXA1-KDM6A-ARHGDIB axis in restraining the malignancy of BCa and identify KDM6A and EZH2 as potential therapeutic targets in the management of BCa.

The impact of culture dimensionality on behavioral epigenetic memory contributing to pluripotent state of iPS cells.

Three-dimensional (3D) culture platforms have been explored to establish physiologically relevant cell culture environment and permit expansion scalability; however, little is known about the mechanisms underlying the regulation of pluripotency of human induced pluripotent stem cells (hiPSCs). This study elucidated epigenetic modifications contributing to pluripotency of hiPSCs in response to 3D culture. Unlike two-dimensional (2D) monolayer cultures, 3D cultured cells aggregated with each other to form ball-like aggregates. 2D cultured cells expressed elevated levels of Rac1 and RhoA; however, Rac1 level was significantly lower while RhoA level was persisted in 3D aggregates. Compared with 2D monolayers, the 3D aggregates also exhibited significantly lower myosin phosphorylation. Histone methylation analysis revealed remarkable H3K4me3 upregulation and H3K27me3 maintenance throughout the duration of 3D culture; in addition, we observed the existence of naïve pluripotency signatures in cells grown in 3D culture. These results demonstrated that hiPSCs adapted to 3D culture through alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and transcriptional expression of pluripotency-associated factors. These results may help design culture environments for stable and high-quality hiPSCs.

Rac1 repression reverses chemoresistance by targeting tumor metabolism.

Tumor metabolism is exemplified by the increased rate of glucose utilization, a biochemical signature of cancer cells. The enhanced glucose hydrolysis enabled by the augmentation of glycolytic flux and the pentose phosphate pathway (PPP) plays a pivotal role in the growth and survival of neoplastic cells. In a recent report, it has been shown that in human breast cancer the GTP binding protein, Rac1 enables resistance to therapy, particularly against the DNA-damaging therapeutics. Significantly, the findings demonstrate that Rac1-dependent chemoresistance involves the upregulation of glycolytic flux as well as PPP. Using multiple approaches, the study demonstrates that disruption of Rac1 activity sensitizes cancer cells to DNA-damaging agents. More importantly, the data uncover a previously unknown PPP regulatory role of Rac1 in breast cancer. Finally, the authors also show the effectiveness and the feasibility of in vivo targeting of Rac1 to enhance the chemosensitivity of breast cancer. This elegant report provokes scientific curiosity to expand our understanding of the intricacies of the role and regulation of Rac1 in cancer.

MiR-485-5p Promotes Neuron Survival through Mediating Rac1/Notch2 Signaling Pathway after Cerebral Ischemia/Reperfusion.

OBJECTIVE: Ischemia-reperfusion (I/R) injury is a pathological feature of ischemic stroke. This study investigated the regulatory role of miR-485-5p in I/R injury. METHODS: SH-SY5Y cells were induced with oxygen and glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury in vitro. Cells were transfected with designated constructs (miR-485- 5p mimics, miR-485-5p inhibitor, lentiviral vectors overexpressing Rac1 or their corresponding controls). Cell viability was evaluated using the MTT assay. The concentrations of lactate dehydrogenase, malondialdehyde, and reactive oxygen species were detected to indicate the degree of oxidative stress. Flow cytometry and caspase-3 activity assay were used for apoptosis assessment. Dual-luciferase reporter assay was performed to confirm that Rac family small GTPase 1 (Rac1) was a downstream gene of miR-485-5p. RESULTS: OGD/R resulted in decreased cell viability, elevated oxidative stress, increased apoptosis, and downregulated miR-485-5p expression in SH-SY5Y cells. MiR-485-5p upregulation alleviated I/R injury, evidenced by improved cell viability, decreased oxidative markers, and reduced apoptotic rate. OGD/R increased the levels of Rac1 and neurogenic locus notch homolog protein 2 (Notch2) signaling-related proteins in cells with normal miR-485-5p expression, whereas miR- 485-5p overexpression successfully suppressed OGD/R-induced upregulation of these proteins. Furthermore, the delivery of vectors overexpressing Rac1 in miR-485-5p mimics-transfected cells reversed the protective effect of miR-485-5p in cells with OGD/R-induced injury. CONCLUSION: This study showed that miR-485-5p protected cells following I/R injury via targeting Rac1/Notch2 signaling suggest that targeted upregulation of miR-485-5p might be a promising therapeutic option for the protection against I/R injury.

Circular RNA RSF1 promotes inflammatory and fibrotic phenotypes of irradiated hepatic stellate cell by modulating miR-146a-5p.

The role of circular RNA (circRNA) in radiation-induced liver disease (RILD) remains largely unknown. In this study, Ras-related C3 botulinum toxin substrate 1 (RAC1) was elevated in irradiated human hepatic stellate cell (HSC) line LX2, the important effector cell mediating RILD. Overexpression of RAC1 promotes cell proliferation, proinflammatory cytokines production, and α-smooth muscle actin expression, which were blocked by microRNA (miR)-146a-5p mimics. CircRNA RSF1 (circRSF1) was upregulated in irradiated LX2 cells and predicted to harbor binding site for miR-146a-5p. Biotinylated-RNA pull down and dual-luciferase reporter detection confirmed the direct interaction of circRSF1 and miR-146a-5p. Enforced expression of circRSF1 increased RAC1 expression by acting as miR-146a-5p sponge to inhibit miR-146a-5p activity, and thus enhanced the cell viability, and promoted inflammatory and fibrotic phenotype of irradiated LX2 cells. These findings indicate a functional regulatory axis composing of circRSF1, miR-146a-5p, and RAC1 in irradiated HSC, which may provide attractive therapeutic targets for RILD.

RAC1 inhibition reverses cisplatin resistance in esophageal squamous cell carcinoma and induces downregulation of glycolytic enzymes.

Development of chemoresistance remains a major challenge in treating esophageal squamous cell carcinoma (ESCC) patients despite treatment advances. However, the role of RAC1 in chemoresistance of ESCC and the underlying mechanisms remain largely unknown. In this study, we found that higher levels of RAC1 expression were associated with poorer prognosis in ESCC patients. Enhanced RAC1 expression increased cell proliferation, migration, and chemoresistance in vitro. Combination therapy using RAC1 inhibitor EHop-016 and cisplatin significantly promoted cell viability inhibition, G2/M phase cycle arrest, and apoptosis when compared to each monotherapy. Mechanistically, glycolysis was significantly downregulated in the RAC1 inhibitor monotherapy group and the combination group via inhibiting AKT/FOXO3a signaling when compared to the control group. Moreover, the silencing of RAC1 inhibited AKT/FOXO3a signaling and cell glycolysis while the upregulation of RAC1 produced an opposite effect. In murine xenograft models, the tumor volume and the expression of glycolytic enzymes were significantly reduced in combination therapy when compared to each monotherapy group. Overall, our study demonstrates that targeting RAC1 with an inhibitor overcomes cisplatin resistance in ESCC by suppressing glycolytic enzymes, which provides a promising strategy for treatment of ESCC in clinical practice.

Distinct contribution of Rac1 expression in cardiomyocytes to anthracycline-induced cardiac injury.

Cardiotoxicity is the dose limiting adverse effect of anthracycline-based anticancer therapy. Inhibitor studies point to Rac1 as therapeutic target to prevent anthracycline-induced cardiotoxicity. Yet, supporting genetic evidence is still missing and the pathophysiological relevance of different cardiac cell types is unclear. Here, we employed a tamoxifen-inducible cardiomyocyte-specific rac1 knock-out mouse model (Rac1flox/flox/MHC-MerCreMer) to investigate the impact of Rac1 expression in cardiomyocytes on cardiac injury following doxorubicin treatment. Distinctive stress responses resulting from doxorubicin treatment were observed, including upregulation of systemic markers of inflammation (IL-6, IL-1α, MCP-1), cardiac damage (ANP, BNP), DNA damage (i.e. DNA double-strand breaks (DSB)), DNA damage response (DDR) and cell death. Measuring the acute doxorubicin response, the serum level of MCP-1 was elevated, cardiac mRNA expression of Hsp70 was reduced and cardiac DDR was specifically enhanced in Rac1 deficient mice. The frequency of apoptotic heart cells remained unaffected by Rac1. Employing a subactue model, the number of doxorubicin-induced DSB was significantly reduced if Rac1 is absent. Yet, the doxorubicin-triggered increase in serum ANP and BNP levels remained unaffected by Rac1. Overall, knock-out of rac1 in cardiomyocytes confers partial protection against doxorubicin-induced cardiac injury. Hence, the data provide first genetic evidence supporting the view that pharmacological targeting of Rac1 is useful to widen the therapeutic window of anthracycline-based anticancer therapy by alleviating acute/subacute cardiomyocyte damage. Furthermore, considering published data obtained from the use of pharmacological Rac1 inhibitors, the results of our study indicate that Rac1-regulated functions of cardiac cell types others than cardiomyocytes additionally influence the adverse outcomes of anthracycline treatment on the heart.

Dendritic spine loss caused by AlCl3 is associated with inhibition of the Rac 1/cofilin signaling pathway.

Aluminum (Al) has neurotoxicity that can result in cognitive dysfunction. Hippocampal dendritic spine loss is a pathological characteristic of cognitive dysfunction. Our previous study reported that Al exposure caused dendritic spine loss in the hippocampus, but the underlying mechanism remains unclear. In this study, rats were orally administered 50, 150 or 450 mg/kg of AlCl3 for 90 days. The dendritic spine density of the CA1 and DG regions was detected by Golgi-Cox staining. The F-actin/G-actin ratio, the expression of drebrin A and the components of the Rac 1/cofilin pathway were measured in the hippocampus. The results obtained showed that AlCl3 caused dendritic spine loss and decreased the F-actin/G-actin ratio. In addition, it was found that AlCl3 downregulated the expression of Rac 1, p-PAK, p-LIMK, p-cofilin and drebrin A and upregulated cofilin expression. Altogether, these results demonstrated that Al inactivated the Rac 1/cofilin pathway by inhibiting the phosphorylation of cofilin and the polymerization of F-actin, resulting in dendritic spine loss in the hippocampus.

The Roc-COR tandem domain of leucine-rich repeat kinase 2 forms dimers and exhibits conventional Ras-like GTPase properties.

The Parkinson's disease (PD)-causative leucine-rich repeat kinase 2 (LRRK2) belongs to the Roco family of G-proteins comprising a Ras-of-complex (Roc) domain followed by a C-terminal of Roc (COR) domain in tandem (called Roc-COR domain). Two prokaryotic Roc-COR domains have been characterized as 'G proteins activated by guanine nucleotide-dependent dimerization' (GADs), which require dimerization for activation of their GTPase activity and bind guanine nucleotides with relatively low affinities. Additionally, LRRK2 Roc domain in isolation binds guanine nucleotides with relatively low affinities. As such, LRRK2 GTPase domain was predicted to be a GAD. Herein, we describe the design and high-level expression of human LRRK2 Roc-COR domain (LRRK2 Roc-COR). Biochemical analyses of LRRK2 Roc-COR reveal that it forms homodimers, with the C-terminal portion of COR mediating its dimerization. Furthermore, it co-purifies and binds Mg2+ GTP/GDP at 1 : 1 stoichiometry, and it hydrolyzes GTP with Km  and kcat  of 22 nM and 4.70 × 10-4  min-1 ,  respectively. Thus, even though LRRK2 Roc-COR forms GAD-like homodimers, it exhibits conventional Ras-like GTPase properties, with high-affinity binding of Mg2+ -GTP/GDP and low intrinsic catalytic activity. The PD-causative Y1699C mutation mapped to the COR domain was previously reported to reduce the GTPase activity of full-length LRRK2. In contrast, this mutation induces no change in the GTPase activity, and only slight perturbations in the secondary structure contents of LRRK2 Roc-COR. As this mutation does not directly affect the GTPase activity of the isolated Roc-COR tandem, it is possible that the effects of this mutation on full-length LRRK2 occur via other functional domains. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Methods to Study the Roles of Rho GTPases in Dendritic Tree Complexity.

Most neurons elaborate a characteristic dendritic arbor which is physiologically important for receiving and processing of synaptic inputs. Pathologically, disturbances in the regulation of dendritic tree complexity are often associated with mental retardation and other neurological deficits. Rho GTPases are major players in the regulation of dendritic tree complexity. They are involved in many signal transduction cascades, activated at the neuronal plasma membrane, and relayed to intracellular proteins that directly rearrange the cytoskeleton. The use of siRNA technology combined with morphometric and imaging techniques allows the roles of individual Rho GTPases, such as Rac1, in dendritic branching to be examined. In this chapter we describe the establishment, transfection, and processing of a primary hippocampal cell culture. Methods to assess the complexity of dendritic arbors like the Sholl analysis, and techniques to investigate Rac1 activity in hippocampal cells, and specifically in neuronal dendrites, such as fluorescence resonance energy transfer (FRET) imaging are presented.

Directional Migration in Esophageal Squamous Cell Carcinoma (ESCC) is Epigenetically Regulated by SET Nuclear Oncogene, a Member of the Inhibitor of Histone Acetyltransferase Complex.

Directional cell migration is of fundamental importance to a variety of biological events, including metastasis of malignant cells. Herein, we specifically investigated SET oncoprotein, a subunit of the recently identified inhibitor of acetyltransferases (INHAT) complex and identified its role in the establishment of front-rear cell polarity and directional migration in Esophageal Squamous Cell Carcinoma (ESCC). We further define the molecular circuits that govern these processes by showing that SET modulated DOCK7/RAC1 and cofilin signaling events. Moreover, a detailed analysis of the spatial distribution of RAC1 and cofilin allowed us to decipher the synergistical contributions of the two in coordinating the advancing dynamics by measuring architectures, polarities, and cytoskeletal organizations of the lamellipodia leading edges. In further investigations in vivo, we identified their unique role at multiple levels of the invasive cascade for SET cell and indicate the necessity for their functional balance to enable efficient invasion as well. Additionally, SET epigenetically repressed miR-30c expression by deacetylating histones H2B and H4 on its promoter, which was functionally important for the biological effects of SET in our cell-context. Finally, we corroborated our findings in vivo by evaluating the clinical relevance of SET signaling in the metastatic burden in mice and a large series of patients with ESCC at diagnosis, observing it's significance in predicting metastasis formation. Our findings uncovered a novel signaling network initiated by SET that epigenetically modulated ESCC properties and suggest that targeting the regulatory axis might be a promising strategy to inhibit migration and metastasis.

High expression of Rac1 is correlated with partial reversed cell polarity and poor prognosis in invasive ductal carcinoma of the breast.

The change of cell polarity is usually associated with invasion and metastasis. Partial reverse cell polarity in IDC-NOS may play a role in lymphatic tumor spread. Rac1 is a kind of polarity related protein. It plays an important role in invasion and metastasis in tumors. We here investigated the expression of Rac1 and partial reverse cell polarity status in breast cancer and evaluated their value for prognosis in breast cancer. The association of the expression of Rac1 and MUC-1 with clinicopathological parameters and prognostic significance was evaluated in 162 cases of IDC-NOS paraffin-embedded tissues by immunohistochemical method. The Rac1 messenger RNA expression was measured by real-time polymerase chain reaction in 30 breast cancer patients, which was divided into two groups of partial reverse cell polarity and no partial reverse cell polarity. We found that lymph node metastasis of partial reverse cell polarity patients was higher than no partial reverse cell polarity patients (Z = -4.030, p = 0.000). Rac1 was upregulated in partial reverse cell polarity group than no partial reverse cell polarity group (Z = -3.164, p = 0.002), and there was correlationship between the expression of Rac1 and partial reverse cell polarity status (rs = 0.249, p = 0.001). The level of Rac1 messenger RNA expression in partial reverse cell polarity group was significantly higher compared to no partial reverse cell polarity group (t = -2.527, p = 0.017). Overexpression of Rac1 and partial reverse cell polarity correlates with poor prognosis of IDC-NOS patients (p = 0.011). Partial reverse cell polarity and lymph node metastasis remained as independent predictors for poor disease-free survival of IDC-NOS (p = 0.023, p = 0.046). Our study suggests that partial reverse cell polarity may lead to poor prognosis of breast cancer. Overexpression of Rac1 may lead to polarity change in IDC-NOS of the breast. Therefore, Rac1 could be a therapeutic target for breast cancer.

Upregulated Vav2 in gastric cancer tissues promotes tumor invasion and metastasis.

Several studies have proved that Vav2 gene is associated with the carcinogenesis of some tumors, but the relationship between Vav2 gene and gastric cancer remains unclear. Purpose of this study is to detect the expression of Vav2 protein in gastric cancer tissues and to evaluate the clinical value of Vav2. Furthermore, both effect of Vav2 gene on invasion and metastasis of gastric cancer cells and its mechanism are investigated in vitro. Results showed that positive rate of Vav2 protein was significantly higher in gastric cancer tissues than in adjacent tissues and notably higher in metastatic lymph nodes than in gastric cancer tissues. Results of western blot were consistent with immunohistochemistry. Expression of Vav2 protein in gastric cancer tissues was related to degree of tumor differentiation, lymph node metastasis, and clinical stages. Inhibition of endogenous Vav2 in BGC823 cells led to significantly decreased cell activity, migration, and invasion ability in vitro, and expression of Rac1, MMP-2, and MMP-9 decreased, whereas expression of TIMP-1 increased. We concluded that Vav2 might promote invasion and metastasis of gastric cancer by regulating some invasion and metastasis-related genes.

Rac1 Pharmacological Inhibition Rescues Human Endothelial Dysfunction.

BACKGROUND: Endothelial dysfunction contributes significantly to the development of vascular diseases. However, a therapy able to reduce this derangement still needs to be identified. We evaluated the effects of pharmacological inhibition of Rac1, a small GTPase protein promoting oxidative stress, in human endothelial dysfunction. METHODS AND RESULTS: We performed vascular reactivity studies to test the effects of NSC23766, a Rac1 inhibitor, on endothelium-dependent vasorelaxation of saphenous vein segments collected from 85 subjects who had undergone surgery for venous insufficiency and from 11 patients who had undergone peripheral vascular surgery. The endothelium-dependent vasorelaxation of the varicose segments of saphenous veins collected from patients with venous insufficiency was markedly impaired and was also significantly lower than that observed in control nonvaricose vein tracts from the same veins. Rac1 activity, reactive oxygen species levels, and reduced nicotine adenine dinucleotide phosphate (NADPH) oxidase activity were significantly increased in varicose veins, and NSC23766 was able to significantly improve endothelium-dependent vasorelaxation of dysfunctional saphenous vein portions in a nitric oxide-dependent manner. These effects were paralleled by a significant reduction of NADPH oxidase activity and activation of endothelial nitric oxide synthase. Finally, we further corroborated this data by demonstrating that Rac1 inhibition significantly improves venous endothelial function and reduces NADPH oxidase activity in saphenous vein grafts harvested from patients with vascular diseases undergoing peripheral bypass surgery. CONCLUSIONS: Rac1 pharmacological inhibition rescues endothelial function and reduces oxidative stress in dysfunctional veins. Rac1 inhibition may represent a potential therapeutic intervention to reduce human endothelial dysfunction and subsequently vascular diseases in various clinical settings.

Rac1 GTPase regulates 11ß hydroxysteroid dehydrogenase type 2 and fibrotic remodeling.

The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11ß hydroxysteroid dehydrogenase type 2 (11ß-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11ß-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11ß-HSD2 expression (r = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11ß-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11ß-HSD2 expression (r = 0.788 and r = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11ß-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11ß-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11ß-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11ß-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling.

Expression of Rac1 alternative 3' UTRs is a cell specific mechanism with a function in dendrite outgrowth in cortical neurons.

The differential expression of mRNAs containing tandem alternative 3' UTRs, achieved by mechanisms of alternative polyadenylation and post-transcriptional regulation, has been correlated with a variety of cellular states. In differentiated cells and brain tissues there is a general use of distal polyadenylation signals, originating mRNAs with longer 3' UTRs, in contrast with proliferating cells and other tissues such as testis, where most mRNAs contain shorter 3' UTRs. Although cell type and state are relevant in many biological processes, how these mechanisms occur in specific brain cell types is still poorly understood. Rac1 is a member of the Rho family of small GTPases with essential roles in multiple cellular processes, including cell differentiation and axonal growth. Here we used different brain cell types and tissues, including oligodendrocytes, microglia, astrocytes, cortical and hippocampal neurons, and optical nerve, to show that classical Rho GTPases express mRNAs with alternative 3' UTRs differently, by gene- and cell- specific mechanisms. In particular, we show that Rac1 originate mRNA isoforms with longer 3' UTRs specifically during neurite growth of cortical, but not hippocampal neurons. Furthermore, we demonstrate that the longest Rac1 3' UTR is necessary for driving the mRNA to the neurites, and also for neurite outgrowth in cortical neurons. Our results indicate that the expression of Rac1 longer 3' UTR is a gene and cell-type specific mechanism in the brain, with a new physiological function in cortical neuron differentiation.

Comparison of neutrophil functions between two strains of inbred mice.

In this study, differences between two strains of inbred mice in aspects of neutrophil function, namely Rac1 expression, chemotaxis, nicotinamide adenine dinucleotide phosphate oxidase activity and formation of neutrophil extracellular traps (NETs), were determined. Neutrophils from CBA/CaH mice exhibited weaker Rac1 expression and a slower chemotactic gradient than BALB/c mice. Furthermore, PMA- or fMLP-stimulated neutrophils from CBA/CaH mice generated much less superoxide and NETs than similarly stimulated neutrophils from BALB/c mice. These findings suggest that neutrophils from BALB/c mice are functionally more efficient than those from CBA/CaH mice.

Rac1 Controls Both the Secretory Function of the Mammary Gland and Its Remodeling for Successive Gestations.

An important feature of the mammary gland is its ability to undergo repeated morphological changes during each reproductive cycle with profound tissue expansion in pregnancy and regression in involution. However, the mechanisms that determine the tissue's cyclic regenerative capacity remain elusive. We have now discovered that Cre-Lox ablation of Rac1 in mammary epithelia causes gross enlargement of the epithelial tree and defective alveolar regeneration in a second pregnancy. Architectural defects arise because loss of Rac1 disrupts clearance in involution following the first lactation. We show that Rac1 is crucial for mammary alveolar epithelia to switch from secretion to a phagocytic mode and rapidly remove dying neighbors. Moreover, Rac1 restricts the extrusion of dying cells into the lumen, thus promoting their eradication by live phagocytic neighbors while within the epithelium. Without Rac1, residual milk and cell corpses flood the ductal network, causing gross dilation, chronic inflammation, and defective future regeneration.

Rac1 as a Potential Pharmacodynamic Biomarker for Thiopurine Therapy in Inflammatory Bowel Disease.

BACKGROUND: Azathioprine and mercaptopurine (MP) are effective in treating patients with inflammatory bowel disease (IBD). Immunosuppressive effects of thiopurines involve T-cell apoptosis after inhibition of GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). This study aimed to assess whether expression and activity of Rac1 or phosphorylated ezrin-radixin-moesin (pERM) in patients with IBD could provide a useful biomarker for the pharmacodynamic thiopurine effect and might be related to clinical effectiveness. METHODS: This was a 2-stage study: stage 1 concerned a cross-sectional cohort of patients with IBD clinically in remission and treated with (n = 10) or without stable weight-based thiopurine therapy (n = 11) and healthy controls (n = 6); stage 2 concerned a prospective study regarding IBD patients with clinically active disease who initiated MP therapy (n = 11) compared with healthy controls (n = 11). Expression and activity of Rac1 and ERM and pERM were determined. RESULTS: The median Rac1 expression was statistically significantly reduced by thiopurine maintenance therapy {0.54 [interquartile range (IQR) 0.47-0.88] versus 0.80 arbitrary units [IQR 0.64-1.46]} compared with patients without immunosuppressive therapy (P = 0.042), but not Rac1 activity and pERM. In responders to MP therapy (n = 6), both median active Rac1 [93 (IQR 81-151) to 76 ng Rac1/mg protein (IQR 62-98)] and Rac1 expression [16.2 (8.8-29.4) to 1.5 arbitrary units (0.9-5.3)] decreased (P = 0.028). In nonresponders (n = 3), Rac1 expression and activity increased. CONCLUSIONS: IBD patients treated with thiopurines had a lower expression of Rac1 compared with those not treated with thiopurine. Effective MP therapy led to decreasing concentrations of Rac1-GTP and Rac1 expression. Therefore, Rac1-GTP and expression of Rac1, but not phosphorylation of ERM, form potentially pharmacodynamic markers of therapeutic thiopurine effectiveness in patients with IBD.