Brian Estevez

Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells

Sexual dimorphisms are typically attributed to the hormonal differences arising once sex differentiation has occurred. However, in some sexually dimorphic diseases that differ in frequency but not severity, the differences cannot be logically connected to the sex hormones. Therefore, we asked whether any aspect of sexual dimorphism could be attributed to chromosomal rather than hormonal differences. Cells taken from mice at d 10.5 postconception (PC) before sexual differentiation, at d 17.5 PC after the first embryonic assertion of sexual hormones, and at postnatal day 17 (puberty) were cultured and exposed to 400 μM ethanol or 20 μM camptothecin or to infection with influenza A virus (multiplicity of infection of 5). The results showed that untreated male and female cells of the same age grew at similar rates and manifested similar morphology. However, they responded differently to the applied stressors, even before the production of fetal sex hormones. Furthermore, microarray and qPCR analyses of the whole 10.5 PC embryos also revealed differences in gene expression between male and female tissues. Likewise, the exposure of cells isolated from fetuses and adolescent mice to the stressors and/or sex hormones yielded expression patterns that reflected chromosomal sex, with ethanol feminizing male cells and masculinizing female cells. We conclude that cells differ innately according to sex irrespective of their history of exposure to sex hormones. These differences may have consequences in the course of sexually dimorphic diseases and their therapy.—Penaloza, C.,Estevez, B., Orlanski, S., Sikorska, M.,Walker, R., Smith, C., Smith, B., Lockshin R.A., Zakeri, Z. Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells. FASEB J. 23, 1869–1879 (2009)

Targeting Integrin and Integrin Signaling in Treating Thrombosis

The critical roles of integrins in thrombosis have enabled the successful development and clinical use of the first generation of integrin antagonists as represented by abciximab (Reopro), eptifibatide (Integrilin), and tirofiban (Aggrastat). These integrin &agr;IIb&bgr;3 antagonists are not only potent antithrombotics but also have significant side effects. In particular, their induction of ligand-induced integrin conformational changes is associated with thrombocytopenia. Increased bleeding risk prevents integrin antagonists from being used at higher doses and in patients at risk for bleeding. To address the ligand-induced conformational changes caused by current integrin antagonists, compounds that minimally induce conformational changes in integrin &agr;IIb&bgr;3 have been developed. Recent studies on the mechanisms of integrin signaling suggest that selectively targeting integrin outside-in signaling mechanisms allows for potent inhibition of thrombosis, while maintaining hemostasis in animal models.

Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis

Objective—Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. Approach and Results—NOX1−/Y platelets showed selective defects in G-protein–coupled receptor–mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2−/− platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1−/Y platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2−/− platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase C&ggr;2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2−/− mice. Wild-type thrombocytopenic mice injected with NOX2−/− platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. Conclusions—NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase C&ggr;2/calcium signaling pathway.

Sex-dependent regulation of cytochrome P450 family members Cyp1a1, Cyp2e1, and Cyp7b1 by methylation of DNA

Sexual differences are only partially attributable to hormones. Cultured male or female cells, even from embryos before sexual differentiation, differ in gene expression and sensitivity to toxins, and these differences persist in isolated primary cells. Male and female cells from Swiss Webster CWF mice manifest sex‐distinct patterns of DNA methylation for X‐ist and for cytochrome P450 (CYP; family members 1a1, 2e1m, and 7b1. Dnmt3l is differentially expressed but not differentially methylated, and Gapdh is neither differentially methylated nor expressed. CYP family genes differ in expression in whole tissue homogenates and cell cultures, with female Cyp expression 2‐ to 355‐fold higher and Dnmt3l 12‐ to 32‐fold higher in males. DNA methylation in the promoters of these genes is sex dimorphic; reducing methylation differences reduces to 1‐ to 6‐fold differences in the expression of these genes. Stress or estradiol alters both methylation and gene expression. We conclude that different methylation patterns partially explain the sex‐based differences in expression of CYP family members and X‐ist, which potentially leads to inborn differences between males and females and their different responses to chronic and acute changes. Sex‐differential methylation may have medical effects.—Penaloza, C.G., Estevez, B., Han, D.M., Norouzi, M., Lockshin, R.A., Zakeri, Z. Sex‐dependent regulation of cytochrome P450 family members Cyp1a1, Cyp2e1, and Cyp7b1 by methylation of DNA. FASEB J. 28, 966–977 (2014). www.fasebj.org

Signaling-mediated cooperativity between glycoprotein Ib-IX and protease-Activated receptors in thrombin-induced platelet activation

Thrombin-induced cellular response in platelets not only requires protease-activated receptors (PARs), but also involves another thrombin receptor, the glycoprotein Ib-IX complex (GPIb-IX). It remains controversial how thrombin binding to GPIb-IX stimulates platelet responses. It was proposed that GPIb-IX serves as a dock that facilitates thrombin cleavage of protease-activated receptors, but there are also reports suggesting that thrombin binding to GPIb-IX induces platelet activation independent of PARs. Here we show that GPIb is neither a passive thrombin dock nor a PAR-independent signaling receptor. We demonstrate a novel signaling-mediated cooperativity between PARs and GPIb-IX. Low-dose thrombin-induced PAR-dependent cell responses require the cooperativity of GPIb-IX signaling, and conversely, thrombin-induced GPIb-IX signaling requires cooperativity of PARs. This mutually dependent cooperativity requires a GPIb-IX-specific 14-3-3-Rac1-LIMK1 signaling pathway, and activation of this pathway also requires PAR signaling. The cooperativity between GPIb-IX signaling and PAR signaling thus drives platelet activation at low concentrations of thrombin, which are important for in vivo thrombosis.

LIM kinase-1 selectively promotes glycoprotein Ib-IX-mediated TXA2 synthesis, platelet activation, and thrombosis

Current antithrombotic drugs have an adverse effect on bleeding, highlighting the need for new molecular targets for developing antithrombotic drugs that minimally affect hemostasis. Here we show that LIMK1(-/-) mice have defective arterial thrombosis in vivo but do not differ from wild-type mice with respect to bleeding time. LIMK1(-/-) mice show a selective defect in platelet activation induced through the von Willebrand Factor (VWF) receptor, the glycoprotein Ib-IX-V complex (GPIb-IX), but not by GPIb-IX-independent platelet agonists. In fact, LIMK1(-/-) platelets show an enhanced reaction to certain GPIb-IX-independent agonists. The defect of LIMK1(-/-) platelets in GPIb-IX-mediated platelet activation is attributed to a selective inhibition in VWF/GPIb-IX-induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and consequent thromboxane A2 (TXA2) production. Supplementing a TXA2 analog, U46619, corrected the defect of LIMK1(-/-) platelets in VWF-induced stable platelet adhesion. Although LIMK1(-/-) platelets also showed reduced actin polymerization after GPIb-IX-mediated platelet aggregation, actin polymerization inhibitors did not reduce TXA2 generation, but rather accelerated platelet aggregation, suggesting that the role of LIMK1 in GPIb-mediated platelet activation is independent of actin polymerization. Thus, LIMK1 plays a novel role in selectively mediating GPIb-IX-dependent TXA2 synthesis and thrombosis and represents a potential target for developing antithrombotic drugs with minimal bleeding side effect.

New concepts and mechanisms of platelet activation signaling

Upon blood vessel injury, platelets are exposed to adhesive proteins in the vascular wall and soluble agonists, which initiate platelet activation, leading to formation of hemostatic thrombi. Pathological activation of platelets can induce occlusive thrombosis, resulting in ischemic events such as heart attack and stroke, which are leading causes of death globally. Platelet activation requires intracellular signal transduction initiated by platelet receptors for adhesion proteins and soluble agonists. Whereas many platelet activation signaling pathways have been established for many years, significant recent progress reveals much more complex and sophisticated signaling and amplification networks. With the discovery of new receptor signaling pathways and regulatory networks, some of the long-standing concepts of platelet signaling have been challenged. This review provides an overview of the new developments and concepts in platelet activation signaling.

The interaction of Gα13 with integrin β1 mediates cell migration by dynamic regulation of RhoA

Gα13 directly binds to the cytoplasmic-domain ExE motif of the integrin β1 subunit. Gα13–β1 interaction mediates β1 integrin–dependent Src activation and transient RhoA inhibition after adhesion. This binding is critical for cell migration on β1 integrin ligands.