Rho-stimulated contractility drives the formation of stress fibers and focal adhesions

Activated rhoA, a ras-related GTP-binding protein, stimulates the appearance of stress fibers, focal adhesions, and tyrosine phosphorylation in quiescent cells (Ridley, A.J., and A. Hall, 1992. Cell. 70:389-399). The pathway by which rho triggers these events has not been elucidated. Many of the agents that activate rho (e.g., vasopressin, endothelin, lysophosphatidic acid) stimulate the contractility of smooth muscle and other cells. We have investigated whether rho's induction of stress fibers, focal adhesions, and tyrosine phosphorylation is the result of its stimulation of contractility. We demonstrate that stimulation of fibroblasts with lysophosphatidic acid, which activates rho, induces myosin light chain phosphorylation. This precedes the formation of stress fibers and focal adhesions and is accompanied by increased contractility. Inhibition of contractility by several different mechanisms leads to inhibition of rho-induced stress fibers, focal adhesions, and tyrosine phosphorylation. In addition, when contractility is inhibited, integrins disperse from focal adhesions as stress fibers and focal adhesions disassemble. Conversely, upon stimulation of contractility, diffusely distributed integrins are aggregated into focal adhesions. These results suggest that activated rho stimulates contractility, driving the formation of stress fibers and focal adhesions and elevating tyrosine phosphorylation. A model is proposed to account for how contractility could promote these events

Rap1b is required for normal platelet function and hemostasis in mice

Rap1b, an abundant small GTPase in platelets, becomes rapidly activated upon stimulation with agonists. Though it has been implicated to act downstream from G protein–coupled receptors (GPCRs) and upstream of integrin αIIbβ3, the precise role of Rap1b in platelet function has been elusive. Here we report the generation of a murine rap1b knockout and show that Rap1b deficiency results in a bleeding defect due to defective platelet function. Aggregation of Rap1b-null platelets is reduced in response to stimulation with both GPCR-linked and GPCR-independent agonists. Underlying the defective Rap1b-null platelet function is decreased activation of integrin αIIbβ3 in response to stimulation with agonists and signaling downstream from the integrin αIIbβ3. In vivo, Rap1b-null mice are protected from arterial thrombosis. These data provide genetic evidence that Rap1b is involved in a common pathway of integrin activation, is required for normal hemostasis in vivo, and may be a clinically relevant antithrombotic therapy target

Tyrosine phosphorylation is involved in reorganization of the actin cytoskeleton in response to serum or LPA stimulation

Tyrosine phosphorylation is known to regulate the formation of focal adhesions in cells adhering to extracellular matrix (ECM). We have investigated the possible involvement of tyrosine phosphorylation and the focal adhesion kinase (FAK) in the cytoskeletal changes induced by serum or lysophosphatidic acid (LPA) in quiescent Swiss 3T3 fibroblasts. As shown previously by others, quiescent cells stimulated with serum or LPA reveal a rapid reappearance of focal adhesions and stress fibers. Here we show that this is accompanied by an increase in phosphotyrosine in focal adhesions and specifically an increase in the tyrosine phosphorylation of FAK. The LPA-stimulated reappearance of focal adhesions and stress fibers is blocked by inhibitors of phospholipase C but not by pertussis toxin (PTX), indicating that this LPA signaling pathway is mediated by phospholipase C activation and does not involve PTX-sensitive G proteins. In the absence of serum or LPA, these cytoskeletal effects and the tyrosine phosphorylation of FAK can be mimicked by sodium orthovanadate in conjunction with hydrogen peroxide, agents that inhibit protein tyrosine phosphatases and thereby elevate levels of phosphotyrosine. Two tyrosine kinase inhibitors, erbstatin and genistein block both the serum-induced tyrosine phosphorylation of FAK and the assembly of focal adhesions and stress fibers. Two other tyrosine kinase inhibitors, tyrphostins 47 and 25, previously shown to inhibit FAK, failed to prevent FAK phosphorylation or the reassembly of focal adhesions and stress fibers in response to serum. However, these inhibitors did prevent FAK phosphorylation and cytoskeletal assembly in response to lysophosphatidic acid (LPA), one component of serum previously shown to stimulate assembly of focal adhesions and stress fibers. Our findings suggest that the response to serum is complex and that although FAK phosphorylation is important, other tyrosine kinases may also be involved

Investments in the Industrial and Logistics Real Estate Sector in Poland Compared to the CEE Countries

Purpose – The aim of this article is to assess Poland’s position and its potential in the light of investments undertaken in the industrial and logistic real estate sector of the Central and Eastern European countries (CEE) as well as to identify the changes contributing to changes within the nature of such investments. Having considered the categories of facilities, supply and demand, also investment capitalization rates and the value of rents the given sector has been subjected to a thorough analysis. Research method – The goal was achieved using quantitative secondary data, also the primary research was conducted among managers using the IDI method. The study was concerned with the trends of changes in the nature of investments in the sector in question in the CEE region in the context of nearshoring, shortening of value chains, and the pandemic. Results – Some mechanisms and determinants, which have been declared important until recently, are insufficient to explain the currently selected options for investments location. The growing popularity of, e.g., q-commerce and the need to provide recipients with the shortest possible delivery time requires the improvement of the supply chain and logistics networks, which is associated with warehouse investments closer to the customer’s location. Originality /value /implications /recommendations – The study concerns an important aspect of investment in service infrastructure. Despite the fact that the Polish commercial real estate market is very popular with foreign investors, mainly from China, the question arises how long Poland will remain the leader in Central and Eastern Europe, taking into account the differences in the capitalization rate or the level of rents in the selected countries.dr Monika WODNICKA: [email protected] hab. Stanisław M. SZUKALSKI: [email protected] Monika WODNICKA - University of Lodzdr hab. Stanisław M. SZUKALSKI - University of LodzBennett N., Lemoine G.J., 2014, What a Difference a Word Makes: Understanding Threats to Performance in a VUCA World, “Business Horizons”, 57(3), pp. 1–7, DOI:10.2139/ssrn.2406676.Coliliers, ExCEEding Borders CEE | 2022, https://docs.colliers.pl/reports/Colliers_Report_ExCEEding_Borders_Industrial_2022.pdf [date of access: 5.08.2022].Cushman, Wakefield, 2021, Polska. Rynek magazynowy II kw. 2021 r.Lipka K., Semaan A., 2021, Raport, Marketbeat, Polska. Rynek magazynowy II kw. 2021 r., Cushman, Wakefield.Lösch A., 1961, Gospodarka przestrzenna, PWE, Warszawa.Polskie Stowarzyszenie Budownictwa Ekologicznego, 2022, Raport, Zrównoważone certyfikowane budynki.PWC, 2022, Perspektywy rozwoju rynku e-commerce w Polsce 2018–2022.Schwab K., 2016, 4IR – Fourth Industrial Revolution, World Economic Forum, Geneva.Schwab K., 2018, Czwarta rewolucja przemysłowa, Wydawnictwo Studio EMKA, Warszawa.Schwab K., P. Vanham, 2022, Kapitalizm interesariuszy. Globalna gospodarka a postęp, ludzie i planeta, Onepress, Gliwice.Smith D.M., 1966, The Theoretical Framework for Geographical Studies of Industrial Location, “Economic Geography”, Vol. 42, No. 2, DOI: abs/10.1080/00130095.1966.11729842.Szukalski S.M., 2021, Ekonomiczno-społeczne skutki czwartej rewolucji przemysłowej (4rp). Wokół serwicyzacji sektora wytwórczego, [in:] A. Stępniak-Kucharska, M. Piekut (eds.) Współczesne problemy gospodarcze gospodarki w czasach kryzysu część I, Płock.Taleb N.N., 2020, Czarny Łabędź. Jak nieprzewidywalne zdarzenia rządzą naszym życiem, Wydawnictwo Zysk i Spółka, Poznań.Weber A.,1909, Uber den Standort der Industrien, Tübingen.Wodnicka M., D. Skurpel, 2021, Growth Global Market of E-Commerce Cross Border The Case of Poland, European Research Studies Journal Volume XXIV, Special Issue 1, pp. 1121–1135 DOI: 10.35808/ersj/2311.Wodnicka M., 2020, Projekty offshoringowe w usługach biznesowych. Pozycja Polski, Wydawnictwo Uniwersytetu Łódzkiego, Łódź.www 1, https://wareh.com/pl [date of access: 30.08.2022].www 2, Axi Immo, https://www.axiimmo.com/raporty-i-publikacje/raport-polski-rynek‑magazynowy-w-i-polowie-2021-r [date of access: 28.08.2022].www 3, Axi Immo, https://www.axiimmo.com/raporty-i-publikacje/ [date of access: 28.08.2022].www 4, https://www.statista.com/statistics/920233/prime-yields-of-warehouses-over-five ‑thousand-square-meter-in-europe-by-city [date of access: 30.08.2022].www 5, Eurostat, https://ec.europa.eu/eurostat/data/database [date of access: 15.09.2022].www 6, JLL, ILL, CEE Investment Market, https://www.jll.pl/content/dam/jll-com/documents/pdf/research/emea/poland/pl/jll-rynek-inwestycyjny-w-europie-srodkowo‑wschodniej-pierwsze-polrocze-2022-r.pdf [date of access: 28.08.2022].www 7, Cushman, Wakefield, https://www.cushmanwakefield.com/pl-pl/poland/insights [date of access: 28.08 2022].www 8, Colliers, Raporty roczne, Market Insights, https://www.colliers.com/plpl/research#sort=%40datez32xpublished%20descending [date of access: 28.08.2022].www 9, https://ecommerce-europe.eu/wp-content/uploads/2021/09/2021-Europe‑an-E-commerce-Report-LIGHT-VERSION.pdf [date of access: 15.09.2022].3(113)11613

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events

Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras.

Vav and Dbl are members of a novel class of oncogene proteins that share significant sequence identity in a approximately 250-amino-acid domain, designated the Dbl homology domain. Although Dbl functions as a guanine nucleotide exchange factor (GEF) and activator of Rho family proteins, recent evidence has demonstrated that Vav functions as a GEF for Ras proteins. Thus, transformation by Vav and Dbl may be a consequence of constitutive activation of Ras and Rho proteins, respectively. To address this possibility, we have compared the transforming activities of Vav and Dbl with that of the Ras GEF, GRF/CDC25. As expected, GRF-transformed cells exhibited the same reduction in actin stress fibers and focal adhesions as Ras-transformed cells. In contrast, Vav- and Dbl-transformed cells showed the same well-developed stress fibers and focal adhesions observed in normal or RhoA(63L)-transformed NIH 3T3 cells. Furthermore, neither Vav- or Dbl-transformed cells exhibited the elevated levels of Ras-GTP (60%) observed with GRF-transformed cells. Finally, GRF, but not Vav or Dbl, induced transcriptional activation from Ras-responsive DNA elements (ets/AP-1, fos promoter, and kappa B). However, like Ras- and GRF-transformed cells, both Vav- and Dbl-transformed cells exhibited constitutively activated mitogen-activated protein kinases (MAPKs) (primarily p42MAPK/ERK2). Since kinase-deficient forms of p42MAPK/ERK2 and p44MAPK/ERK1 inhibited Dbl transformation, MAPK activation may be an important component of its transforming activity. Taken together, our observations indicate that Vav and Dbl transformation is not a consequence of Ras activation and instead may involve the constitutive activation of MAPKs

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated α 2 β 1 activation

The platelet α2β1 integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that α2β1 function can be activated via inside-out signaling, similar to the prototypical platelet integrin αIIbβ3. However, signaling molecules that regulate α2β1 activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates αIIbβ3 activation

Functional redundancy between RAP1 isoforms in murine platelet production and function

RAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we conditionally deleted Rap1a and/or Rap1b in the megakaryocytic lineage (mKO). Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired pro- platelet formation by megakaryocytes. In platelets, RAP isoforms had both redundant and isoform-ˇspecific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α- granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood-lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. While important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation