Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.

Os objetivos desse estudo foram: 1) Padronizar a pesquisa do anti-tTg, comparando-o com o anticorpo antiendomísio (AAE) e 2) Avaliar as interações da tTg com a fibronectina. 49 celíacos e 124 controles com AAE negativo foram avaliados. O AAE foi pesquisado por imunofluorescência indireta e a reatividade contra a tTg e a fibronectina por ELISA in house e com kits comerciais. O antitTg foi positivo em 46,9% e 100% dos celíacos com o ELISA in house e com kits comerciais, respectivamente. A adição de fibronectina não melhorou a sensibilidade do ELISA. Em conclusão: a detecção do antitTg por ELISA apresenta percentual elevado de falso-positivos, não podendo substituir a pesquisa do AAE / The aims of the current study were: to standardize the detection of anti-tTg antibodies, comparing them with antiendomysial antibodies (EMA) and to assess the interaction of tTg with fibronectin. 49 celiac patients and 124 controls were enrolled. EMA was detected by indirect immunofluorescence reaction and tTg and fibronectin reactivity by in house ELISA and with commercially available kits. Seropositivity to anti-tTG was found in 46.9% and 100% of patients by the in house technique and by commercial kits, respectively. Fibronectin addition did not improve the ELISA sensibility. In conclusion, ELISA for anti-tTG detection has a high rate of false positive results and does not replace EMA

Integrins are heterodimers that mediate cell adhesion in many physiological processes. Binding of integrins to ligands provides anchorage and signals for the cell. However, how force regulates integrin/ligand dissociation is unclear. Atomic force microscopy was used to measure the force dependence of lifetimes of single bonds between a FN fragment and integrin α₅β₁. First, lifetime-force relationships demonstrated that force prolonged bond lifetimes in the 10-30 pN range, a behavior called catch bonds. Changing divalent cations from Ca²⁺/Mg²⁺ to Mg²⁺/EGTA and to Mn²⁺ caused more pronounced catch bonds. A truncated α₅β₁ construct containing the headpiece but not the legs (trα₅β₁-Fc) formed much longer-lived catch bonds in the same force range. Bindings of two activating mAbs, 12G10 and TS2/16, left shift the catch bond and converted catch bonds to slip bonds, respectively. Catch bonds may provide a mechanical mechanism for the cell to regulate adhesion by applying different forces. Second, FNIII₇₋₁₀/α₅β₁-Fc/GG-7 bond was stretched to ~ 30 pN and then relaxed to ~ 7 pN at which the bond's lifetime was measured. The strong bond state induced by the 30 pN stretching stayed stable even after the force was reduced to 7 pN. In other words, lower the force would not weaken FNIII₇₋₁₀/α₅β₁-Fc bond once it had been stretched. Similar behaviors were observed for FNIII₇₋₁₀/trα₅β₁-Fc and FNIII₇₋₁₀/mα₅β₁interactions. In addition, the efficiency of the force to induce such a strong bond state for FNIII₇₋₁₀/α₅β₁-Fc interaction in 2 mM Mg²⁺/EGTA condition was characterized. The probability of force to induce the strong bond state increased as force increased and when the force reached 26 pN, all bonds were transit to the strong state. Moreover, reversible unbending of α₅β₁binding with FNIII₇₋₁₀ under mechanical force were observed, which proved that integrin bending and unbending was dynamic. Importantly, integrin could restore bent conformation even when engaged with its ligand, providing a mechanism for mechanotransduction. Third, structural changes of α₅β₁under force were observed. The structural changes did not change the trend of lifetime-force relationships of FNIII₇₋₁₀/α₅β₁/GG-7 bond. Moreover, the lifetime for the structural changes to occur and molecular length changes caused by them were characterized.

Epithelial to mesenchymal transition (EMT), the transdifferentation of an epithelial cell into a mesenchymal fibroblast, is a cellular process necessary for embryonic development and wound healing. However, uncontrolled EMT can result in accumulation of myofibroblasts and excessive deposition of ECM, contributing to the pathological progression of fibrotic diseases such as pulmonary fibrosis. The ability to control EMT is important for development of novel therapeutics for fibrotic pathologies and for designing novel biomaterials for tissue engineering applications seeking to promote EMT for development of complex tissues. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin-mediated interactions with extracellular matrix (ECM) proteins and soluble growth factors such as TGFβ. TGFβ, a potent inducer of EMT, is activated via cell contraction-mediated mechanical release of the growth factor from a macromolecular latency complex. Thus TGFβ activity and subsequent EMT may be influenced by the biochemical and biophysical state of the surrounding ECM. Based on these knowns, it was hypothesized that both changes in integrin engagement and increases in substrate rigidity would modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin-specific interactions with fibronectin (Fn) fragments displaying both the RGD and PHSRN binding sites facilitate cell binding through α5β1 and α3β1 integrins, and lead to maintenance of epithelial phenotype, while Fn fragments displaying only the RGD site facilitate cell binding through αv integrins and lead to EMT. An in depth investigation into α3β1 binding to Fn fragments indicates that binding is dependent on both the presence and orientation of the PHSRN site. Studies investigating the contribution of ECM stiffening on EMT responses show that increasingly rigid Fn substrates are sufficient to induce spontaneous EMT. Analysis of TGFβ-responsive genes implicate TGFβ-expression, activation or signaling as a mechanism for the observed EMT responses. Together these results suggest that the ECM micromechanical environment is a significant contributor to the onset of EMT responses and provide insights into the design of biomaterial-based microenvironments for the control of epithelial cell phenotype.

As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes can induce osteoblast proliferation in vitro, but do so only when direct cell-to-cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of megakaryocytes with another cell-type of mesenchymal origin - the fibroblast. Our findings implicate the involvement of fibronectin/RGD-binding integrins including á3â1 (VLA-3) and á5â1 (VLA-5) as well as glycoprotein IIb (CD41), all of which are known to be expressed on megakaryocyte membranes. Furthermore, we demonstrate that IL-3 can enhance megakaryocyte-induced osteoblast activation in vitro, as demonstrated in the megakaryocyte-fibroblast model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic-mesenchymal cell activation are mechanistically analogous.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2007. / Radhakrishna, Harish, Committee Member ; Zhu, Cheng, Committee Member ; LaPlaca, Michelle C., Committee Member ; Garca, Andrs J., Committee Chair ; Kowalczyk, Andrew P., Committee Member.

Os objetivos desse estudo foram: 1) Padronizar a pesquisa do anti-tTg, comparando-o com o anticorpo antiendomísio (AAE) e 2) Avaliar as interações da tTg com a fibronectina. 49 celíacos e 124 controles com AAE negativo foram avaliados. O AAE foi pesquisado por imunofluorescência indireta e a reatividade contra a tTg e a fibronectina por ELISA in house e com kits comerciais. O antitTg foi positivo em 46,9% e 100% dos celíacos com o ELISA in house e com kits comerciais, respectivamente. A adição de fibronectina não melhorou a sensibilidade do ELISA. Em conclusão: a detecção do antitTg por ELISA apresenta percentual elevado de falso-positivos, não podendo substituir a pesquisa do AAE / The aims of the current study were: to standardize the detection of anti-tTg antibodies, comparing them with antiendomysial antibodies (EMA) and to assess the interaction of tTg with fibronectin. 49 celiac patients and 124 controls were enrolled. EMA was detected by indirect immunofluorescence reaction and tTg and fibronectin reactivity by in house ELISA and with commercially available kits. Seropositivity to anti-tTG was found in 46.9% and 100% of patients by the in house technique and by commercial kits, respectively. Fibronectin addition did not improve the ELISA sensibility. In conclusion, ELISA for anti-tTG detection has a high rate of false positive results and does not replace EMA

With over 50,000 deaths and 80,000 disorders annually in the United States resulting from traumatic brain injury (TBI), there is a demand for improved therapeutic strategies. Cell transplantation offers the potential to treat TBI by targeting multiple mechanisms in a sustained fashion. However, efforts are needed to improve survival and integration of transplanted cells, and ultimately enhance functional recovery. Using tissue engineering strategies, we aimed to mimic key aspects of fetal tissue grafts by combining neural stem cells with a fibronectin or laminin based scaffold that could be delivered to the injured brain in a minimally invasive fashion. We found that the incorporation of extracellular matrix proteins into a cell transplantation paradigm led to improved donor cell survival and restored cognitive ability for treated animals. To begin to examine how fibronectin and laminin mediate these improvements, we first examined the endogenous role of these two proteins in the injured brain. Using a clinically-relevant model of TBI, we found both proteins are increased in the injured brain at acute time points. The spatial localization of fibronectin and laminin with specific support cells in the brain suggests a role for these proteins in repair, warranting further investigation. Using conditional plasma fibronectin knockout animals, we found that fibronectin is neuroprotective to the traumatically injured brain. Specifically, injured fibronectin knockout animals had more severe motor and cognitive deficits, increased cell death, and decreased retention of phagocytic cells compared to injured wild type animals. Thus, we have identified novel therapeutic treatments for TBI which utilize tissue engineered transplants and/or exploit endogenous repair mechanisms for fibronectin.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.