Natalia V. Bogatcheva

Molecular Mechanisms of Thrombin-Induced Endothelial Cell Permeability

Confluent endothelium serves as a selective barrier between the vascular space of blood vessels and underlying tissues. Compromised barrier function of the endothelium in response to inflammation mediators, such as thrombin, is accompanied by reversible cell rounding and interendothelial gap formation. Endothelial barrier integrity substantially depends on the cytoskeleton, which ensures actin stress fiber formation and via actomyosin-driven contraction regulates cell shape and adhesion. Recent studies have shown the sequence of events that mediate signal transduction in endothelial cells. Binding of thrombin with its receptor initiates activation of heterotrimeric G-proteins, which, in turn, entails a decrease in cAMP level in the cell, increase in intracellular Ca2+ and diacylglycerol concentration, and activation of the small G-protein Rho. Phosphorylation of myosin light chains as a result of activation of myosin light chain kinase and inactivation of myosin phosphatases stimulates stress fiber formation and triggers actomyosin contraction. In addition, thrombin-induced rearrangement in the endothelial cytoskeleton is regulated by Ca2+/calmodulin-dependent protein kinase, protein kinase C, and tyrosine protein kinases. This review focuses on presently known biochemical mechanisms of cell response to thrombin and their role in endothelial barrier dysfunction.

Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins.

The modern classification of small heat shock proteins (sHsp) is presented and peculiarities of their primary structure and the mechanism of formation of oligomeric complexes are described. Data on phosphorylation of sHsp by different protein kinases are presented and the effect of phosphorylation on oligomeric state and chaperone activity of sHsp is discussed. Intracellular location of sHsp under normal and stress conditions is described and it is emphasized that under certain condition sHsp interact with different elements of cytoskeleton. The literature concerning the effect of sHsp on polymerization of actin in vitro is analyzed. An attempt is made to compare effects of sHsp on polymerization of actin in vitro with the results obtained on living cells under normal conditions and after heat shock or hormone action. The literature concerning possible effects of sHsp on cell motility is also analyzed.

The role of cytoskeleton in the regulation of vascular endothelial barrier function.

The cytoskeleton is vital to the function of virtually all cell types in the organism as it is required for cell division, cell motility, endo- or exocytosis and the maintenance of cell shape. Endothelial cells, lining the inner surface of the blood vessels, exploit cytoskeletal elements to ensure the integrity of cell monolayer in quiescent endothelium, and to enable the disintegration of the formed barrier in response to various agonists. Vascular permeability is defined by the combination of transcellular and paracellular pathways, with the latter being a major contributor to the inflammation-induced barrier dysfunction. This review will analyze the cytoskeletal elements, which reorganization affects endothelial permeability, and emphasize signaling mechanisms with barrier-protective or barrier-disruptive potential.

Relaxin Promotes Prostate Cancer Progression

Purpose: To understand the role of relaxin peptide in prostate cancer, we analyzed the expression of relaxin and its receptor in human prostate cancer samples, the effects of relaxin signaling on cancer cell phenotype in vitro, and the effects of increased serum relaxin concentrations on cancer progression in vivo. Experimental Design: The relaxin and its receptor leucine-rich repeat containing G protein–coupled receptor 7 (LGR7) expression were studied by quantitative reverse transcription-PCR (11 benign and 44 cancer tissue samples) and by relaxin immunohistochemistry using tissue microarrays containing 10 normal and 69 cancer samples. The effects of relaxin treatment and endogenous relaxin/LGR7 suppression via short interfering RNA in PC-3 and LNCaP cells were analyzed in vitro. The effect of transgenic relaxin overexpression [Tg(Rln1)] on cancer growth and survival was evaluated in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP). Results: The relaxin mRNA expression was significantly higher in recurrent prostate cancer samples. In tissue microarrays of the 10 normal tissues, 8 had low staining in epithelial cells, whereas only 1 of 9 high-grade prostatic intraepithelial neoplasia lesions had low expression (P = 0.005) and only 29 of 65 cancers had low expression (P = 0.047). Stimulation with relaxin increased cell proliferation, invasiveness, and adhesion in vitro. The suppression of relaxin/LGR7 via short interfering RNAs decreased cell invasiveness by 90% to 95% and growth by 10% to 25% and increased cell apoptosis 0.6 to 2.2 times. The Tg(Rln1) TRAMP males had shorter median survival time, associated with the decreased apoptosis of tumor cells, compared with non-Tg(Rln1) TRAMP animals. Conclusions: Relaxin signaling plays a role in prostate cancer progression.

The role of relaxin in endometrial cancer

Relaxin (RLN) is a naturally occurring hormone that is known to modulate connective tissue remodeling in the uterus and cervix. Our goal was to investigate the role of RLN in endometrial cancer. RLN expression was evaluated using immunohistochemistry in 57 samples of invasive endometrial carcinoma (EC) and 10 benign endometria. 67% of high-stage (III/IV) tumors demonstrated strong RLN expression compared to 37% of low-stage (I/II) cases. Strong RLN expression associated significantly with high-grade and depth of myometrial invasion. Notably, strong RLN expression was associated with a significantly shorter overall survival (p

Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability.

Up to date, the nature of the sepsis‐induced vascular leakage is understood only partially, which limits pharmacological approaches for its management. Here we studied the protective effect of cAMP using endotoxin‐induced hyperpermeability as a model for barrier dysfunction observed in gram‐negative sepsis. We demonstrated that the alleviation of lipopolysaccharide (LPS)‐induced barrier compromise could be achieved by the specific activation of either protein kinase A (PKA) or Epac with cAMP analogs Bnz‐cAMP or O‐Me‐cAMP, respectively. We next studied the involvement of PKA substrates VASP and filamin1 in barrier maintenance and LPS‐induced barrier compromise. Depletion of both VASP and filamin1 with the specific siRNAs significantly exacerbated both the quiescent cells barrier and LPS‐induced barrier dysfunction, suggesting barrier‐protective role of these proteins. VASP depletion was associated with the more severe loss of ZO‐1 peripheral staining in response to LPS, whereas filamin1‐depleted cells reacted to LPS with more robust stress fiber induction and more profound changes in ZO‐1 and VE‐cadherin peripheral organization. Both VASP and filamin1 phosphorylation was significantly increased as a result of PKA activation. We next analyzed the effect of VASP and filamin1 depletion on the PKA‐dependent alleviation of LPS‐induced barrier compromise. We observed that Bnz‐cAMP ability to counteract LPS‐induced hyperpermeability was attenuated only by VASP, but not filamin1 depletion. Our data indicate that while PKA‐dependent VASP phosphorylation contributes to the protective effect of cAMP elicited on LPS‐compromised monolayers, filamin1 phosphorylation is unlikely to play a significant role in this process. J. Cell. Physiol. 221: 750–759, 2009.

Mitogen-Activated Protein Kinases in Endothelial Pathophysiology

Endothelial cells continuously respond to extracellular stimuli such as chemical signals produced by circulating blood elements or mechanical forces such as shear stress. Proinflammatory cytokines, mitogens, reactive oxygen species, and shear stress trigger signal molecules to initiate multiple intracellular pathways, which often converge at mitogen-activated protein (MAP) kinase activation. The MAP kinase superfamily represents a burgeoning area of clinical investigation for treatment of various inflammatory and oncologic diseases and plays an essential role in mediating response to infection, ischemia/reperfusion injury, and vessel healing and remodeling through regulation of such diverse phenomena as endothelial cell proliferation, migration, apoptosis, and endothelial barrier function. The downstream effects of MAP kinase activation include modulation of gene expression via up-regulation of various transcription factors. In addition to these sustained effects, MAP kinases coordinate more immediate responses that affect dynamic cytoskeletal rearrangements necessary for cell migration and regulation of barrier function. This review discusses the important regulatory roles of MAP kinases in the vital physiologic functions of endothelium, focusing mainly on the role of MAP kinases in the maintenance of endothelial barrier.

INSL3/LGR8 role in testicular descent and cryptorchidism

Cryptorchidism, generally referred to a failure of testicular descent into the scrotum, is the most frequent (up to 3-4% at birth) congenital anomaly in newborn boys. Cryptorchidism is closely associated with impaired fertility, and represents an established risk factor for testicular cancer. Like other genital defects, cryptorchidism is believed to be caused by either endocrine or genetic abnormalities, or both. Recent elucidation of the molecular mechanism of the rodent testicular descent, and, in particular, the critical role of Insl3 (insulin-like 3) and its receptor Great/Lgr8 encouraged the search for naturally occurring mutations in the human homologues of these genes in the affected patient population. Genetic analysis revealed several functionally deleterious mutations in both INSL3 and GREAT/LGR8 genes. However, although some of mutations were found only in cryptorchid patients, it remains to be verified whether there is a causative link between the presence of mutations in INSL3 or GREAT/LGR8 and the undescended testis phenotype in men. The data and analysis of published studies indicate that mutations in these two genes might account for only a small portion of all cases of this disease in the human population.

Role of tyrosine kinase signaling in endothelial cell barrier regulation

Phosphorylation of proteins on tyrosine acts as a reversible and specific trigger mechanism, forming or disrupting regulatory connections between proteins. Tyrosine kinases and phosphatases participate in multiple cellular processes, and considerable evidence now supports a role for tyrosine phosphorylation in vascular permeability. A semipermeable barrier between the vascular compartment and the interstitium is maintained by the integrity of endothelial monolayer, controlling movement of fluids, macromolecules and leucocytes. Barrier function is regulated by the adjustment of paracellular gaps between endothelial cells (ECs) by two antagonistic forces, centripetal cytoskeletal tension and opposing cell-cell and cell-matrix adhesion forces. Both cytoskeletal filaments and adhesion sites are intimately linked in complex machinery which is regulated by multiple signaling events including protein phosphorylation and/or protein translocation to specific intracellular positions. Tyrosine kinases occupy key positions in the mechanism controlling cell responses mediated through various cell surface receptors, which use tyrosine phosphorylation to transduce extracellular signal.

Developmental Expression and Gene Regulation of Insulin-like 3 Receptor RXFP2 in Mouse Male Reproductive Organs

Abstract The mutations of testicular insulin-like 3 (INSL3) hormone or its receptor RXFP2 cause cryptorchidism in male mice. Here we have examined Rxfp2 gene expression at different stages of embryonic and postnatal mouse development in male reproductive tissues employing quantitative RT-PCR and several RXFP2-specific antibodies directed toward different parts of the RXFP2 protein. Receptor expression was markedly increased after birth and was readily detectable in the epididymis, Leydig cells, and germ cells of the testis. The strongest expression was detected in adult mouse cremaster muscle. INSL3 treatment increased cell proliferation of embryonic gubernacular and TM3 embryonic Leydig cells, implicating active INSL3-mediated autocrine signaling in these cells and identifying TM3 as a novel in vitro model to study the effects of RXFP2 signaling. We generated Tg(Rxfp2-cre)Aia (Rxfp2-iCre) transgenic mice expressing improved Cre recombinase (iCre) under the control of the 2.4-kb mouse Rxfp2 promoter. The iCre was expressed in the gubernacular ligament at E14.5, indicating that this promoter is able to drive Rxfp2 gene expression during transabdominal testis descent. We demonstrated that the transcription factor Sox9, a known male sex determination factor, is expressed in mouse embryonic gubernacula and upregulated human, but not mouse, promoter luciferase reporter constructs. In conclusion, we have determined the developmental expression profile of INSL3 receptor employing newly characterized RXFP2 antisera and a novel Rxfp2-iCre transgenic mouse model. We determined the promoter region capable of providing the gubernacular-specific expression of Rxfp2. Analysis of RXFP2 promoter identified SOX9 as a new transcriptional enhancer of human gene expression.