Matrix Dynamics Group

Permanent URI for this collectionhttps://hdl.handle.net/1807/16910

The Matrix Dynamics Group was established in 1973 with funding from the Medical Research Council of Canada, now known as Canadian Institutes of Health Research. Originally the group focused on the regulation of cells from the periodontium and was known as the MRC group in Periodontal Physiology. The group currently consists of 5 principal investigators and focuses on the regulation of connective tissue matrix proteins in health and disease. The Group's major strength is in bringing a full spectrum of scientific and technical experience to the issues of matrix remodeling and the signaling networks that control remodeling.

News

Matrix Dynamics Group website

Browse

Now showing 1 - 20 of 58

Nonactivated versus Thrombin-Activated Platelets on Wound Healing and Fibroblast-to-Myofibroblast Differentiation In Vivo and In Vitro
(Lippincott, Williams & Wilkins, 2012-01) Scherer, S. S.; Tobalem, M.; Vigato, E.; Heit, Y.; Modarressi, A.; Hinz, Boris; Pittet, B.; Pietramaggiori, G.
BACKGROUND: Platelet preparations for tissue healing are usually preactivated before application to deliver concentrated growth factors. In this study, the authors investigated the differences between nonactivated and thrombin-activated platelets in wound healing. METHODS: The healing effects (i.e., wound closure, myofibroblast formation, and angiogenesis) of nonactivated and thrombin-activated platelets were compared in experimental wounds in diabetic (db/db) animals. In vitro, fibroblast phenotype and function were tested in response to platelets and activated platelets. No treatment served as a negative control. RESULTS: Wounds treated with platelets reached 90 percent closure after 15 days, faster than activated platelets (26 days), and with higher levels of myofibroblasts and angiogenesis. In vitro, platelets enhanced cell migration and induced two-fold higher myofibroblast differentiation and contraction compared with activated platelets. CONCLUSIONS: Platelets stimulate wound healing more efficiently compared with activated platelets by enhancing fibroblast differentiation and contractile function. Similar levels of growth factors may induce different biological effects when delivered "on demand" rather than in an initial bolus.
An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells
(Nature Publishing Group, 2010-04) Lee, W. Y.; Moriarty, T. J.; Wong, C. H.; Zhou, H.; Strieter, R. M.; van Rooijen, N.; Chaconas, G.; Kubes, P.
Here we investigate the dynamics of the hepatic intravascular immune response to a pathogen relevant to invariant natural killer T cells (iNKT cells). Immobilized Kupffer cells with highly ramified extended processes into multiple sinusoids could effectively capture blood-borne, disseminating Borrelia burgdorferi, creating a highly efficient surveillance and filtering system. After ingesting B. burgdorferi, Kupffer cells induced chemokine receptor CXCR3-dependent clustering of iNKT cells. Kupffer cells and iNKT cells formed stable contacts via the antigen-presenting molecule CD1d, which led to iNKT cell activation. An absence of iNKT cells caused B. burgdorferi to leave the blood and enter the joints more effectively. B. burgdorferi that escaped Kupffer cells entered the liver parenchyma and survived despite Ito cell responses. Kupffer cell-iNKT cell interactions induced a key intravascular immune response that diminished the dissemination of B. burgdorferi.
Perioperative use of non-steroidal anti-inflammatory drugs might impair dental implant osseointegration
(Wiley, 2014-09-30) Winnett, Brent; Tenenbaum, Howard C.; Ganss, Ben; Jokstad, Asbjørn
OBJECTIVE: To appraise whether adverse biological events following oral implant placement may be associated with perioperative use of non-steroidal anti-inflammatory drugs (NSAIDs). METHODS: All patients treated in a university faculty postgraduate dental clinic between 1979 and 2012 that had experienced a failing and surgically removed dental implant (292 implants in 168 patients) were contacted to solicit additional information about their present dental and medical status and frequency of current and past use of NSAIDs. Potential associations between perioperative NSAIDs use and the occurrence of adverse biological events were explored by the use of 2 × 2 tables and two-tailed Fisher's exact tests. RESULTS: One hundred and four patients with initially 468 implants had experienced 238 implant failures, of which 197 were due to failing osseointegration (42%). Sixty of the participants, initially with 273 implants, had used NSAIDs perioperatively and experienced 44% implant failures, versus 38% in the non-NSAID cohort. The NSAID cohort experienced 3.2 times more cases of radiographic bone loss greater than 30% of the vertical height of their remaining implants and 1.9 times more cases of cluster failures, defined as failure of 50% or more of the implant(s) placed. CONCLUSIONS: Notwithstanding that a retrospective study design is open to potential bias, the current data indicate that dental implant osseointegration may be affected negatively by an inhibitory effect of NSAIDs on bone healing in vulnerable patients. Future and better clinical studies than the current should be designed to appraise more precisely the potential effects of NSAIDs on implant osseointegration in study populations that are not limited by stringent medical inclusion and exclusion criteria.
Healing of periodontal tissues following transplantation of cells in a rat orthodontic tooth movement model
(Edward H. Angle Society of Orthodontists, 2008-09) Nayak, B. N.; Wiltshire, W. A.; Ganss, B.; Tenenbaum, H.; McCulloch, C. H.; Lekic, C.
OBJECTIVE: To determine the fate and differentiation of transplanted periodontal ligament (PL) precursor cells and mouse embryonic stem (ES) cells and their relative capacity to regenerate wounded periodontium. MATERIALS AND METHODS: Orthodontic tooth movement was introduced 24 hours before transplantation of PL or ES cells, and rats were euthanized either 24 hours or 72 hours after cell transplantation. The control rats received either no tooth movement and no cell transplantation or tooth movement and no cell transplantation. Differentiation of transplanted cells was assessed from mandibular periodontal histological tissue sections by immunohistochemical methods using monoclonal antibodies against PL cell differentiation markers. Data were analyzed using Student's t-test at a significance level of P = .05. RESULTS: Transplantation of PL and ES cells resulted in a higher number of osteopontin, bone sialoprotein, and alpha-smooth muscle actin labeled transplanted cells, predominantly around the blood vessels of the periodontium in study rats compared with control rats (cell transplantation but no orthodontic tooth movement, P = .05). Combined treatments of tooth movement and cell transplantation resulted in enhanced regeneration of the periodontium as a result of tooth movement. Transplantation of PL cells induced a higher number of differentiating cells in the PL and alveolar bone than did transplantation of ES cells. CONCLUSIONS: Orthodontic tooth movement promotes the differentiation of transplanted cells, and the differentiation occurs predominantly in the paravascular areas of the periodontium. In terms of regeneration of wounded periodontium, transplantation of PL cells produced a higher level of regeneration than ES cells, possibly because of PL cell plasticity and the capacity to undergo effective differentiation in the periodontal cellular microenvironment.
Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia
(National Academy of Sciences, 2014-09-23) Lee, W. Y.; Sanz, M. J.; Wong, C. H.; Hardy, P. O.; Salman-Dilgimen, A.; Moriarty, T. J.; Chaconas, G.; Marques, A.; Krawetz, R.; Mody, C. H.; Kubes, P.
CXCR6-GFP(+) cells, which encompass 70% invariant natural killer T cells (iNKT cells), have been found primarily patrolling inside blood vessels in the liver. Although the iNKT cells fail to interact with live pathogens, they do respond to bacterial glycolipids presented by CD1d on liver macrophage that have caught the microbe. In contrast, in this study using dual laser multichannel spinning-disk intravital microscopy of joints, the CXCR6-GFP, which also made up 60-70% iNKT cells, were not found in the vasculature but rather closely apposed to and surrounding the outside of blood vessels, and to a lesser extent throughout the extravascular space. These iNKT cells also differed in behavior, responding rapidly and directly to joint-homing pathogens like Borrelia burgdorferi, which causes Lyme disease. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted dissemination attempts by these microbes into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway, an observation also made in vitro for iNKT cells from joint but not liver or spleen. These results suggest a novel, critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier and explains a large increase in pathogen burden of B. burgdorferi in the joint of iNKT cell-deficient mice, and perhaps the greater susceptibility of humans to this pathogen because of fewer iNKT cells in human joints.
Activation of antibacterial autophagy by NADPH oxidases
(National Academy of Sciences, 2009-04-14) Huanga, Ju; Canadiena, Veronica; Lama, Grace Y.; Steinberga, Benjamin E.; Dinauer, Mary C.; Magalhaes, Marco A. O.; Glogauer, Michael; Grinstein, Sergio
Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcγ receptors (FcγRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy.
The impact of the ovarian microenvironment on the anti-tumor effect of SPARC on ovarian cancer
(NRC Research Press, 2012-02) Greenaway, J. B.; Koehler, A.; McCulloch, Christopher A.; Petrik, J.; Brown, T. J.; Ringuette, M. J.
A lack of host-derived SPARC promotes disease progression in an intraperitoneal (IP) ID8 mouse model of epithelial ovarian cancer (EOC). Since orthotopic injection (OT) of ID8 cells better recapitulates high-grade serous cancer, we examined the impact of host-derived SPARC following OT injection. Sparc(-/-) and wild-type (WT) mice were injected with ID8 cells either OT or IP and tumors were analyzed at the moribund stage. Sparc(-/-) mice had reduced survival and fewer well-defined abdominal lesions compared with WT controls after IP injection, whereas no differences were observed in survival or abdominal lesions between Sparc(-/-) and WT mice after OT injection. No differences in mass or collagen content were observed in ovarian tumors between OT-injected Sparc(-/-) and WT mice. The abdominal wall of the IP-injected Sparc(-/-) mice exhibited immature and less abundant collagen fibrils compared with WT mice both in injected and non-injected controls. In contrast to human EOC, SPARC was expressed by the tumor cells but was absent in reactive stroma of WT mice. Exposure to the ovarian microenvironment through OT injections alters the metastatic behaviour of ID8 cells, which is not affected by the absence of host-derived SPARC.
Glycated collagen induces a11 integrin expression through TGF-ß2 and Smad3
(Wiley, 2014-06-24) Talior-Volodarsky, I.; Arora, P. D.; Wang, Y.; Zeltz, C.; Connelly, K. A.; Gullberg, D.; McCulloch, Christopher A.
The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the a11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal-glycated collagen regulates a11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased a11 integrin and a-smooth muscle actin expression. Compared with native collagen, binding of purified a11b1 integrin to glycated collagen was reduced by fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF-b2 but not TGF-b1 or TGF-b3. The increased expression of TGF-b2 was inhibited by triple helical collagen peptides that mimic the a11b1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with a11 integrin luciferase promoter constructs, glycated collagen activated the a11 integrin promoter. Analysis of a11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between 809 and 1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF-b2 signaling pathway that stimulates a11 integrin expression through Smad2/3 binding elements in the a11 integrin promoter, which is important for myofibroblast formation and fibrosis.
Epithelial-specific knockout of the Rac1 gene leads to enamel defects
(Wiley, 2011-12) Huang, Z.; Kim, J.; Lacruz, R. S.; Bringas, P. Jr.; Glogauer, Michael; Bromage, T. G.; Kaartinen, V. M.; Snead, M. L.
The Ras-related C3 botulinum toxin substrate 1 (Rac1) gene encodes a 21-kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction, activities with consequences for cell growth, adhesion, migration, and differentiation. To examine the role(s) played by RAC1 protein in cell-matrix interactions and enamel matrix biomineralization, we used the Cre/loxP binary recombination system to characterize the expression of enamel matrix proteins and enamel formation in Rac1 knockout mice (Rac1(-/-)). Mating between mice bearing the floxed Rac1 allele and mice bearing a cytokeratin 14-Cre transgene generated mice in which Rac1 was absent from epithelial organs. Enamel of the Rac1 conditional knockout mouse was characterized by light microscopy, backscattered electron imaging in the scanning electron microscope, microcomputed tomography, and histochemistry. Enamel matrix protein expression was analyzed by western blotting. Major findings showed that the Tomes' processes of Rac1(-/-) ameloblasts lose contact with the forming enamel matrix in unerupted teeth, the amounts of amelogenin and ameloblastin are reduced in Rac1(-/-) ameloblasts, and after eruption, the enamel from Rac1(-/-) mice displays severe structural defects with a complete loss of enamel. These results support an essential role for RAC1 in the dental epithelium involving cell-matrix interactions and matrix biomineralization.
Impact of matrix metalloproteinases on inhibition of mineralization by fetuin
(Wiley, 2013-06) Schure, R.; Costa, K. D.; Rezaei, R.; Lee, W.; Laschinger, C; Tenenbaum, H. C.; McCulloch, Christopher A.
BACKGROUND AND OBJECTIVE: Human subjects affected by inflammatory diseases, such as periodontitis, may be at increased risk for the development of cardiovascular diseases and calcification of atheromas; however, the potential mechanisms have not been defined. Alpha-2-Heremans Schmid glycoprotein (fetuin A) is an abundant serum glycoprotein of ~49 kDa that inhibits ectopic arterial calcification. We examined whether matrix metalloproteinases (MMPs), which are increased in inflammatory diseases, including periodontitis, bind and degrade fetuin and alter its ability to inhibit calcification in vitro. MATERIAL AND METHODS: Binding and cleavage of fetuin by MMPs were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-silico analyses and mass spectrometry. The effects of intact and MMP-degraded human fetuin on mineralization were measured in a cell-free assay. RESULTS: From in-silico analyses and literature review, we found that only MMP-3 (stromelysin) and MMP-7 (matrilysin) were predicted to cleave human fetuin at levels that were physiologically relevant. In-vitro assays showed that MMP-7, and, to a lesser extent, MMP-3, degraded human fetuin in a time- and dose-dependent manner. Fetuin peptides generated by MMP-7 cleavage were identified and sequenced by mass spectrometry; novel cleavage sites were found. Hydroxyapatite mineralization in vitro was strongly inhibited by fetuin (> 1 μm), as was MMP-3-cleaved fetuin, while MMP-7-cleaved fetuin was threefold less effective in blocking mineralization. CONCLUSION: MMP-7 and, to a lesser extent, MMP-3, affect the ability of fetuin to inhibit the formation of hydroxyapatite in vitro. These data suggest that the MMPs increased in inflammatory diseases, such as periodontitis, could affect regulation of mineralization and potentially enhance the risk of calcified atheroma formation.
Culture of primary bovine chondrocytes on a continuously expanding surface inhibits dedifferentiation
(Mary Ann Liebert, 2012-12-18) Rosenzweig, D. H.; Matmati, M.; Khayat, G.; Chaudhry, S.; Hinz, Boris; Quinn, T. M.
Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and α-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies.
It has to be the av: myofibroblast integrins activate latent TGF-ß1
(Nature Publishing Group, 2013-12-05) Hinz, Boris
Cell-mediated activation of latent TGF-β1 is a key promoting event in fibrosis in all organs. A new study shows that specific targeting of the αv subunit of integrins in fibrogenic myofibroblasts effectively reduces developing and established fibrosis in liver, kidneys and lungs (pages 1617–1624).
Tumor Cell Invasion Is Promoted by Interstitial Flow-Induced Matrix Priming by Stromal Fibroblasts.
(American Association for Cancer Research, 2011-02) Shieh, Adrian C. ; Rozansky, Hallie A. ; Hinz, Boris ; Swartz, Melody A.
Interstitial flow emanates from tumors into the microenvironment where it promotes tumor cell invasion. Fibroblasts are key constituents of the tumor stroma which modulate the mechanical environment by matrix remodeling and contraction. Here we explore how interstitial fluid flow affects fibroblast-tumor cell interactions. Using a 3-D invasion assay and MDA-MB-435S cells co-cultured with dermal fibroblasts in a collagen matrix, we demonstrated a synergistic enhancement of tumor cell invasion by fibroblasts in the presence of interstitial flow. Interstitial flow also drove transforming growth factor (TGF)-β1 and collagenase-dependent fibroblast migration, consistent with previously described mechanisms where flow promotes invasion through autologous chemotaxis and increased motility. Concurrently, migrating fibroblasts enhanced tumor cell invasion by matrix priming via Rho-mediated contraction. We propose a model where interstitial flow promotes fibroblast migration through TGF-β1 and collagenase activity, positioning fibroblasts to locally reorganize collagen fibers via Rho-dependent contractility, enhancing tumor cell invasion via mechanotactic cues. This represents a novel mechanism where interstitial flow causes stromal remodeling that facilitates tumor invasion.
Flaxseed enhances the beneficial effect of low-dose estrogen therapy at reducing bone turnover and preserving bone microarchitecture in ovariectomized rats.
(NRC Research Press (Canadian Science Publishing), 2014-01) Sacco, SM ; Chen, J ; Ganss, Bernhard ; Thompson, LU ; Ward, WE
Our previous research showed greatest protection to vertebral bone mineral density and strength in ovariectomized (OVX) rats when lignan- and alpha-linolenic acid-rich flaxseed (FS) is combined with low-dose estrogen therapy (LD) compared to either treatment alone. This study determined the effects of combined FS+LD on serum and tissue markers of bone turnover and microarchitecture to explain our previous findings. 3-month-old OVX rats were randomized to negative control (NEG), FS, LD or FS+LD for 2 or 12 weeks, meaningful time points for determining effects on markers of bone metabolism and bone structure, respectively. Ground FS was added to the AIN-93M diet (100g/kg diet) and LD (0.42μg 17β-estradiol/kg body weight/day) was delivered by subcutaneous implant. Sham rats were included as positive control. Bone formation (e.g. osteocalcin), bone resorption (e.g. tartrate-resistant acid phosphatase-5β (TRAP-5β)), as well as osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed from the 2-week study by commercial assays (serum) and/or histology (vertebra). Vertebral bone microarchitecture was measured from the 12-week study using microcomputed tomography. In serum, FS+LD and LD induced lower TRAP-5β and osteocalcin, and higher OPG and OPG/RANKL ratio versus NEG and FS (p<0.05). In vertebrae, FS+LD induced higher OPG and lower osteocalcin versus NEG (p<0.01) and did not differ from LD and FS. FS+LD improved bone microarchitecture versus NEG, FS and LD (p<0.05). In conclusion, FS+LD protects bone tissue due to a reduction in bone turnover. However, elucidating the distinctive action of FS+LD on bone turnover compared to LD requires further investigation.
Comparative Temporospatial Expression Profiling of Murine Amelotin Protein during Amelogenesis.
(Karger, 2012-05) Somogyi-Ganss, E ; Nakayama, Y ; Iwasaki, K ; Nakano, Y ; Stolf, Diana ; McKee, MD ; Ganss, B
Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization.
Force-induced apoptosis mediated by the Rac/PAK/p38 signaling pathway is regulated by filamin A.
(Portland Press, 2012-07) Shifrin, Y ; Pinto, VI ; Hassanali, A ; Arora, PD ; McCulloch, Christoper A
Cells in mechanically challenged environments cope with high amplitude exogenous forces that can lead to cell death but the mechanisms that mediate force-induced apoptosis and the identity of mechanoprotective cellular factors are not defined. We assessed apoptosis in 3T3 and HEK cells exposed to tensile forces applied through !1 integrins. Apoptosis was mediated by Racdependent activation of p38". Depletion of Pak1, a downstream effector of Rac, prevented forceinduced p38 activation and apoptosis. Rac was recruited to sites of force transfer by filamin A, which inhibited force-induced apoptosis mediated by Rac and p38". We conclude that in response to tensile force, filamin A regulates Rac-dependent signals, which induce apoptosis through Pak1 and p38.
Ras-guanine nucleotide releasing factors 1 and 2 interact with PLC? at focal adhesions to enable IL-1-induced Ca2+ signaling, ERK activation and MMP-3 expression.
(Portland Press, 2013-02) Wang, Qin ; Siminovitch, KA ; Downey, GP ; McCulloch, CA
Interleukin-1 (IL-1) signaling in anchorage-dependent cells involves focal adhesion-restricted and Ca2+-dependent Ras and extracellular signal-related kinase (ERK) activation that lead to MMP release and extracellular matrix remodeling. Ras activity is regulated in part by the Ca2+-responsive Ras guanine-nucleotide-releasing factors 1 and 2 (Ras-GRF1/2), but the mechanisms that link and localize IL-1-induced Ca2+ signaling to focal adhesions are not defined. Herein we characterized the role of Ras-GRF1/2 in Ca2+ and Ras􀵺ERK signaling after IL-1 stimulation.
Filamin A protects cells against force-induced apoptosis by stabilizing talin- and vinculin-containing cell adhesions.
(Federation of American Society of Experimental Biology (FASEB), 2014-01) Pinto, VI ; Senini, VW ; Wang, Y ; Kazembe, MP ; McCulloch, MC
In mechanically loaded tissues such as weight-bearing joints, myocardium and periodontal ligament, pathophysiological forces can disrupt cell-matrix contacts, which in turn induce cell death leading to tissue and organ dysfunction. Protection against force-induced cell death may be mediated by filamin A (FLNa), an actinbinding protein that regulates β1 integrin-mediated cell adhesion. We examined collagen distribution, cell densities and cell death in periodontal ligament of filamin A wildtype and fibroblast conditional-knockout mice. Deletion of FLNa in fibroblasts was associated with 2-fold increase of acellular areas in the periodontal ligament and a 7-fold higher proportion of apoptotic cells. In cultured fibroblasts with FLNa knockdown, we examined the impact of supra-physiological forces (1 pN/μm2 cell area; applied through the β1 integrin) on recruitment of talin and vinculin to focal adhesions and on apoptosis. Compared with wildtype, FLNa knockdown cells exhibited 3-fold increases of floating cells after overnight force application and a 2-fold increase of cell detachment. Force induced time-dependent reductions (p<0.05) of the numbers of activated β1 integrin, talin and vinculin-stained adhesions in FLNa knockdown compared with wildtype cells. We conclude that FLNa protects against apoptosis in cells subjected to mechanical forces and this protection is mediated by enhanced formation and maturation of matrix adhesions.
Aquaporin 9 phosphorylation mediates membrane localization and neutrophil polarization.
(Society for Leukocyte Biology, 2011-11) Karlsson, T ; Glogauer, Michael ; Ellen, EP ; Loitto, VM ; Magnusson, KE ; es, MA Magalhã
Neutrophils are of prime importance in the host innate defence against invading microorganisms by employing two primary mechanisms, locomotion towards and phagocytosis of the prey. Recent research points to pivotal roles for water channels known as aquaporins in cell motility. Here, we focused on the role of aquaporin 9 (AQP9) in chemoattractant-induced polarization and migration of primary mouse neutrophils and neutrophil-like HL60 cells. We found that AQP9 is phosphorylated downstream of the fMLF receptor or PMA stimulation in primary human neutrophils. The dynamics of AQP9 was assessed using GFP-tagged AQP9 constructs and other fluorescent markers through various live-cell imaging techniques. Expression of wild-type or the phosphomimic S11D AQP9 changed cell volume regulation as a response to hyperosmotic changes and enhanced neutrophil polarization and chemotaxis. Wt AQP9 and S11D AQP9 displayed a very dynamic distribution at the cell membrane, whereas the phosphorylation-deficient S11A AQP9 failed to localize to the plasma membrane. Furthermore, we found that Rac1 regulated the translocation of AQP9 to the plasma membrane. Our results show that AQP9 plays an active role in neutrophil volume regulation and migration. The display of AQP9 at the plasma membrane depends on AQP9 phosphorylation, which appeared to be regulated through a Rac1-dependent pathway.
The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils.
(Society for Leukocyte Biology, 2009-12) Tole, S ; Mukovozov, IM ; Huang, YW ; Magalhaes, MA ; Yan, M ; Crow, MR ; Liu, GW ; Sun, CX ; Durocher, Y ; Glogauer, Michael ; Robinson, Lisa A
In inflammatory diseases circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not effectively prevent neutrophil recruitment. The Slit family of secreted proteins, and their transmembrane receptor, Roundabout (Robo), repel axonal migration during central nervous system development. Emerging evidence shows that by inhibiting the activation of Rho-family GTPases, Slit2/Robo also inhibit migration of other cell types towards a variety of chemotactic factors, in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow, and detected Robo-1 expression. Using video-microscopic live cell tracking, we found that Slit2 selectively impaired directional migration, but not random movement, of neutrophils towards formyl-methionyl-leucyl-phenylalanine (fMLP). Slit2 also inhibited neutrophil migration towards other chemoattractants, namely C5a and interleukin (IL)-8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant-induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2, but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and macrophage inflammatory protein-2 (MIP-2). In all instances, Slit2 effectively reduced neutrophil recruitment (p < 0.01). Collectively, these data demonstrate that Slit2 potently inhibits chemotaxis, but not random motion, of circulating neutrophils, and point to Slit2 as a potential new therapeutic for preventing localized inflammation.

Browse

Recent Submissions