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Activated Cdc42‐associated kinase 1 (ACK1), a widely expressed nonreceptor tyrosine kinase, is often amplified in cancer and has been shown to interact with Cell division cycle 42 (Cdc42), Epidermal growth factor receptor (EGFR), and several other cancer‐relevant molecules, suggesting a possible role for ACK1 in development and tumor formation. To directly address this scenario, we generated mice lacking a functional ACK1 gene (ACK1 ko) using CRISPR genome editing. ACK1 ko mice developed normally, displayed no obvious defect in tissue maintenance, and were fertile. Primary ACK1‐null keratinocytes showed normal phosphorylation of EGFR, but a tendency toward reduced activation of AKT serine/threonine kinase 1 (Akt) and Mitogen‐activated protein kinase 1 (Erk). DMBA/TPA‐induced skin tumor formation did not reveal significant differences between ACK1 ko and control mice. Deletion of the ACK1 gene in the breast cancer cell lines MDA‐MB‐231, 67NR, MCF7, 4T1, and T47D caused no differences in growth. Furthermore, EGF‐induced phosphorylation kinetics of Erk, Akt, and p130Cas were not detectably altered in T47D cells by the loss of ACK1. Finally, loss of ACK1 in MDA‐MB‐231 and T47D breast cancer cells had a very limited or no effect on directed cell migration. These data do not support a major role for ACK1 in Cdc42 and EGFR signaling, development, or tumor formation. [ABSTRACT FROM AUTHOR]

Abstract: The Rho GTPase Rac1 is a multifunctional protein involved in distinct pathways ranging from development to pathology. The aim of the present study was to unravel the contribution of neuronal Rac1 in regulating the response to brain injury induced by permanent focal cerebral ischemia (pMCAO). Our results show that pMCAO significantly increased total Rac1 levels in wild type mice, mainly through rising nuclear Rac1, while a reduction in Rac1 activation was observed. Such changes preceded cell death induced by excitotoxic stress. Pharmacological inhibition of Rac1 in primary neuronal cortical cells prevented the increase in oxidative stress induced after overactivation of glutamate receptors. However, this was not sufficient to prevent the associated neuronal cell death. In contrast, RNAi‐mediated knock down of Rac1 in primary cortical neurons prevented cell death elicited by glutamate excitotoxicity and decreased the activity of NADPH oxidase. To test whether in vivo down regulation of neuronal Rac1 was neuroprotective after pMCAO, we used tamoxifen‐inducible neuron‐specific conditional Rac1‐knockout mice. We observed a significant 50% decrease in brain infarct volume of knockout mice and a concomitant increase in HIF‐1α expression compared to littermate control mice, demonstrating that ablation of Rac1 in neurons is neuroprotective. Transmission electron microscopy performed in the ischemic brain showed that lysosomes in the infarct of Rac1‐ knockout mice were preserved at similar levels to those of non‐infarcted tissue, while littermate mice displayed a decrease in the number of lysosomes, further corroborating the notion that Rac1 ablation in neurons is neuroprotective. Our results demonstrate that Rac1 plays important roles in the ischemic pathological cascade and that modulation of its levels is of therapeutic interest. [ABSTRACT FROM AUTHOR]

Keratinocytes, in response to irritants, secrete pro-inflammatory mediators which recruit and activate immune and mesenchymal cells, including fibroblasts, to repair the skin. Fibroblasts respond by synthesising collagen and promoting the crosslinking extracellular matrix (ECM). We recently showed that the deletion of Rac1 in keratinocytes causes heightened inflammation due to aberrant crosstalk with immune cells. Indeed, the skin of these mice shows a higher inflammatory response to the induction of irritant contact dermatitis (ICD), and also even to treatment with a vehicle alone, compared with controls. As inflammation is intimately linked with fibrotic disease in the skin, this raised the question as to whether this deletion may also affect the deposition and arrangement of the dermal ECM. This study assessed the effects of Rac1 deletion in keratinocytes and of the heightened inflammatory status by induction of ICD on the tissue localisation and arrangements of dermal collagen. Qualitative analysis did not reveal evidence for the formation of pathologies in the dermis. However, quantitative analysis did reveal some perturbations in the dermal matrix, namely that only the combination of the lack of Rac1 and ICD affects the architectural organisation of the dermal collagen, and that a higher inflammatory state in the tissue (i.e. when Rac1 is deleted in the keratinocytes or ICD is induced in the skin, or a combination of both) influences the diameter of the collagen fibrils. It is proposed that this increase in the diameter of collagen fibrils due to inflammation may serve as pre-fibrotic marker enabling earlier determination of fibrosis and earlier treatment. This study has revealed previously unknown effects on the ECM due to the deletion of Rac1 in keratinocytes. [ABSTRACT FROM AUTHOR]

Cytoskeletal reorganization is essential to keratinocyte function. Rac1 regulates cytoskeletal reorganization through signaling pathways such as the cofilin cascade. Cofilin severs actin filaments after activation by dephosphorylation. Rac1 was knocked out in mouse keratinocytes and it was found that actin filaments disassembled. In the epidermis of mice in which Rac1 was knocked out only in keratinocytes, cofilin phosphorylation was aberrantly elevated, corresponding to repression of the phosphatase slingshot1 (SSH1). These effects were independent of the signaling pathways for p21-activated kinase/LIM kinase (Pak/LIMK), protein kinase C, or protein kinase D or generation of reactive oxygen species. Similarly, when actin polymerization was specifically inhibited or Rac1 was knocked down, cofilin phosphorylation was enhanced and SSH1 was repressed. Repression of SSH1 partially blocked actin depolymerization induced by Rac1 depletion. Therefore, aberrant cofilin phosphorylation that induces actin polymerization might be a consequence of actin disassembly induced by the absence of Rac1. © 2015 BioFactors, 41(5):352-359, 2015 [ABSTRACT FROM AUTHOR]

Key points The role of the small G-protein Rac1 was investigated in smooth muscle, using a smooth muscle-specific knockout mouse and pharmacological blockers., Inhibition of the interaction between Rac1 and upstream regulators inhibited the α-receptor contractions and potentiated prostaglandin F2α contractions in vascular tissue., The inhibition was mediated via an attenuation of the Ca2+ transient., A global inhibition of Rac1 activity inhibited contractions in response to several agonists in a range of smooth muscle tissues., The results demonstrate a novel Rac1-associated signalling pathway for regulation of smooth muscle contraction., Abstract The role of the small GTP-binding protein Rac1 in smooth muscle contraction was examined using small molecule inhibitors (EHT1864, NSC23766) and a novel smooth muscle-specific, conditional, Rac1 knockout mouse strain. EHT1864, which affects nucleotide binding and inhibits Rac1 activity, concentration-dependently inhibited the contractile responses induced by several different modes of activation (high-K+, phenylephrine, carbachol and protein kinase C activation by phorbol-12,13-dibutyrate) in several different visceral (urinary bladder, ileum) and vascular (mesenteric artery, saphenous artery, aorta) smooth muscle tissues. This contractile inhibition was associated with inhibition of the Ca2+ transient. Knockout of Rac1 (with a 50% loss of Rac1 protein) lowered active stress in the urinary bladder and the saphenous artery consistent with a role of Rac1 in facilitating smooth muscle contraction. NSC23766, which blocks interaction between Rac1 and some guanine nucleotide exchange factors, specifically inhibited the α1 receptor responses (phenylephrine) in vascular tissues and potentiated prostaglandin F2α and thromboxane (U46619) receptor responses. The latter potentiating effect occurred at lowered intracellular [Ca2+]. These results show that Rac1 activity is required for active contraction in smooth muscle, probably via enabling an adequate Ca2+ transient. At the same time, specific agonists recruit Rac1 signalling via upstream modulators, resulting in either a potentiation of contraction via Ca2+ mobilization (α1 receptor stimulation) or an attenuated contraction via inhibition of Ca2+ sensitization (prostaglandin and thromboxane receptors). [ABSTRACT FROM AUTHOR]

Rho GTPases are a family of small GTPases, which play an important role in the regulation of the actin cytoskeleton. Not surprisingly, Rho GTPases are crucial for cell migration and therefore highly important for cancer cell invasion and the formation of metastases. In addition, Rho GTPases are involved in growth and survival of tumor cells, in the interaction of tumor cells with their environment, and they are vital for the cancer supporting functions of the tumor stroma. Recent research has significantly improved our understanding of the regulation of Rho GTPase activity, the specificity of Rho GTPases, and their function in tumor stem cells and tumor stroma. This review summarizes these novel findings and tries to define challenging questions for future research. © 2013 BioFactors, 40(2):226-235, 2014 [ABSTRACT FROM AUTHOR]

Rhoh is a hematopoietic system-specific GTPase. Rhoh-deficient T cells have been shown to have a defect in TCR signaling manifested during their thymic development. Our aims were to investigate the phenotype of peripheral Rhoh-deficient T cells and to explore in vivo the potential benefit of Rhoh deficiency in a clinically relevant situation, in which T-cell inhibition is desirable. In murine allogenic kidney transplantation, Rhoh deficiency caused a significant 75% reduction of acute and chronic transplant rejection accompanied by 75% lower alloantigen-specific antibody levels and significantly better graft function. This effect was independent of the lower T-cell numbers in Rhoh-deficient recipients, because injection of equal numbers of Rhoh-deficient or control T cells into kidney transplanted mice with SCID led again to a significant 60% reduction of rejection. Mixed lymphocyte reaction revealed that the weaker alloreactivity was associated with a 85% lower cytotoxicity and a 50-80% lower cytokine release in Rhoh-deficient T cells without an influence on the secretion itself. Antigen uptake and presentation in DC were unaffected by Rhoh deficiency. These findings stress the importance of Rhoh for the function of peripheral T cells. [ABSTRACT FROM AUTHOR]

To study the role of Cdc42 in the establishment of epithelial polarity during mammalian development, we generated murine Cdc42-null embryonic stem cells and analyzed peri-implantation development using embryoid bodies (EBs). Mutant EBs developed endoderm and underlying basement membrane, but exhibited defects of cell polarity, cell-cell junctions, survival, and cavitation. These defects corresponded to a decreased phosphorylation and membrane localization of aPKC, a reduced phosphorylation of GSK3β, and a diminished activity of Rac1. However, neither Rac1 nor the kinase function of GSK3β seem to contribute to cell polarization and cell-cell contacts. In contrast, EBs expressing dominant-negative (dn) PKCζ mimicked well the phenotype of Cdc42-null EBs, suggesting a major role of aPKC in mediating cell polarization downstream of Cdc42. Finally, aggregation experiments with endodermal cell lines suggested that Cdc42 might affect formation of adherens and tight junctions by PKCζ-dependent regulation of the protein levels of p120 catenin and E-cadherin. Developmental Dynamics 236:2767-2778, 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

β integrins are thought to play a central role in maintaining lymphocytes within mucosal epithelia via their interactions with extracellular matrix proteins and subepithelial cellular components within and underlying the basement membrane. In the current study type a (CD8αβTCRαβ) and type b (CD8ααTCRγδ and CD8ααTCRαβ) intraepithelial lymphocyte (IEL) subsets within the mouse small intestine were found to express functional β integrin and the β integrin α chain partners α, α, and α. Using inducible β integrin-knockout bone marrow-chimeric mice we demonstrate that IEL expression of α and α but not α is dependent on expression of the β chain. Importantly, deletion of the β chain in IEL did not alter the number or composition of lymphocytes within the intestinal epithelium. Thus, while IEL express functional β integrins, these are not required to maintain lymphocytes within intestinal epithelia. This result is discussed in the light of conventional views of intestinal lymphocyte homing and localization. [ABSTRACT FROM AUTHOR]

Integrin receptors connect the extracellular matrix to the actin cytoskeleton. This interaction can be viewed as a cyclical liaison, which develops again and again at new adhesion sites only to cease at sites of de-adhesion. Recent work has demonstra [ABSTRACT FROM AUTHOR]

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin α2β1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of β1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of β1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, β1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s-1) and high (1000 s-1) shear flow conditions, probably through binding of αIIbβ3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including α2β1 integrin, which strengthens adhesion without being essential. [ABSTRACT FROM AUTHOR]

β1 integrins are ubiquitously expressed receptors that mediate cell-cell and cell-extracellular matrix interactions. To analyze the function of β1 integrin in skin we generated mice with a keratinocyte-restricted deletion of the β1 integrin gene using the cre-loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of α6β4 integrin, and the number of helnideslnosolnes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin-5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal-epidermal junction. In contrast, the integrity of the basement membrane surrounding the β1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of β1 integrins in hair follide morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiaton and proliferation, and in the formation and/or maintenance of hemidesmosomes. [ABSTRACT FROM AUTHOR]

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating -helical and -type structure, and by protease resistance and immunological analysis. It was highly modified by N- and O-glycosylation but not by glycosaminoglycans. Ultra- centrifugation showed non-covalent association into oligomers with molar masses of 1000–1500 kDa. Electron microscopy showed ring-like shapes with diameters of 30–40 nm. M2BP bound in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin β1 subunits but not to α2 and α6 subunits, RGD peptide or lactose. This distinguishes cell adhesion of M2BP from that of laminin and excludes involvement of lactose-binding galectin-3. Immunological assays demonstrated variable secretion by cultured human cells of M2BP, which was detected in the extracellular matrix of several mouse tissues. [ABSTRACT FROM AUTHOR]

Dopamine‐producing tyrosine hydroxylase (TH) neurones in the hypothalamic arcuate nucleus (ARC) have recently been shown to be involved in ghrelin signalling and body weight homeostasis. In the present study, we investigate the role of the intracellular regulator RhoA in hypothalamic TH neurones in response to peripheral hormones. Diet‐induced obesity was found to be associated with increased phosphorylation of TH in ARC, indicating obesity‐associated increased activity of ARC TH neurones. Mice in which RhoA was specifically knocked out in TH neurones (TH‐RhoA−/− mice) were more sensitive to the orexigenic effect of peripherally administered ghrelin and displayed an abolished response to the anorexigenic hormone leptin. When TH‐RhoA−/− mice were challenged with a high‐fat high‐sucrose (HFHS) diet, they became hyperphagic and gained more body weight and fat mass compared to wild‐type control mice. Importantly, lack of RhoA prevented development of ghrelin resistance, which is normally observed in wild‐type mice after long‐term HFHS diet feeding. Patch‐clamp electrophysiological analysis demonstrated increased ghrelin‐induced excitability of TH neurones in lean TH‐RhoA−/− mice compared to lean littermate control animals. Additionally, increased expression of the orexigenic hypothalamic neuropeptides agouti‐related peptide and neuropeptide Y was observed in TH‐RhoA−/− mice. Overall, our data indicate that TH neurones in ARC are important for the regulation of body weight homeostasis and that RhoA is both a central effector in these neurones and important for the development of obesity‐induced ghrelin resistance. The obese phenotype of TH‐RhoA−/− mice may be a result of increased sensitivity to ghrelin and decreased sensitivity to leptin, resulting in increased food intake. [ABSTRACT FROM AUTHOR]

Focuses on the use of integrin blockade as therapy in chronic inflammation. Background on the process of leukocyte extravasation, where the integrin receptors can contribute to the rolling of leukocytes on the endothelium and are essential for the firm adhesion of leukocytes to the vessel wall; Role of integrin in the transmigration through the endothelium; Integrins expressed by all leukocytes; Ability of integrins to bind to endothelial receptors such as vascular cell adhesion molecule-1 and intercellular cell adhesion molecules to extracellular matrix molecules such as laminins and collagens, but also to other proteins such as the inactivated complement factor iC3b or bacterial polysaccharides. Importance of integrins as signaling receptors which after ligand binding initiate a variety of signaling cascades; Details of a study that demonstrated a remarkable protective effect of a4 integrin-blocking antibodies in patients with relapsing multiple sclerosis.