Distribution by Scientific Domains

Selected Abstracts

Greater growth hormone and insulin response in women than in men during repeated bouts of sprint exercise

M. Esbjörnsson
Abstract Aim:, In a previous study, sprint training has been shown to increase muscle cross-sectional area in women but not in men [Eur J Appl Physiol Occup Physiol 74 (1996) 375]. We hypothesized that sprint exercise induces a different hormonal response in women than in men. Such a difference may contribute to explaining the observed gender difference in training response. Method:, Metabolic and hormonal response to three 30-s sprints with 20-min rest between the sprints was studied in 18 physically active men and women. Results:, Accumulation of blood lactate [interaction term gender (g) × time (t): P = 0.022], and plasma ammonia (g × t: P < 0.001) after sprint exercise was greater in men. Serum insulin increased after sprint exercise more so in women than in men (g × t: P = 0.020), while plasma glucose increased in men, but not in women (g × t: P < 0.001). Serum growth hormone (GH) increased in both women and men reaching similar peak levels, but with different time courses. In women the peak serum GH level was observed after sprint 1, whereas in men the peak was observed after sprint 3 (g × t; P < 0.001). Serum testosterone tended to decrease in men and increase in women (g × t: P = 0.065). Serum cortisol increased approx. 10,15% after sprint exercise, independent of gender (time: P = 0.005). Conclusion:, Women elicited a greater response of serum GH and insulin to sprint exercise. This may contribute to explaining the earlier observed muscle hypertrophy in women in response to sprint training. [source]

Lithium and KB-R7943 effects on mechanics and energetics of rat heart muscle

P. Bonazzola
ABSTRACT The role of calcium influx on energy expenditure during cardiac contraction was studied. For this purpose, the described ability of lithium and KB-R 7943 (KBR) to diminish Ca entry through Na,Ca exchanger (Ponce-Hornos & Langer, J Mol Cell Cardiol 1980, 12, 1367, Satoh et al., Circulation 2000, 101, 1441) were used. In isolated contractions (contractions elicited after at least 5 min of rest) LiCl 45 mmol L,1 decreased pressure developed and pressure,time integral from 42.3 ± 2.7 and 14.5 ± 1.2 to 32.1 ± 3.4 mN mm,2 and 8.3 ± 0.9 mN mm,2 s, respectively. A similar effect was observed in regular contractions (at 0.16 Hz stimulation). The presence of KBR (5 ,mol L,1) in the perfusate induced a slight but not significant decrease in pressure developed and pressure,time integral in steady-state contractions. As it was previously described, the heat involved in a heart muscle contraction can be decomposed into several components (H1, H2, H3 and H4), but only one (H3) was associated with force generation. While H3 decreased with lithium in both types of contractions, H3/PtI ratio remained unaltered, indicating that the economy for pressure maintenance was unaffected. To further investigate the role of Ca entry on force development, a condition in which the contraction is mainly dependent on extracellular calcium was studied. An ,extra' stimulus applied 200 ms after the regular one in a muscle stimulated at 0.16 Hz induces a contraction with this characteristic (Marengo et al., Am J Physiol 1999, 276, H309). Lithium induced a strong decrease in pressure,time integral and H3 associated with this contraction (43 and 45%, respectively) with no change in H3/PtI ratio. Lithium also reduced (53%) an energy component (H2) associated with Ca cycling. The use of KBR showed qualitatively similar results [i.e. a 33% reduction in pressure,time integral associated with the extrasystole (ES) with no changes in H3/PtI ratio and a 30% reduction in the H2 component]. Li and KBR effects appear to be additive and in the presence of 45 mmol L,1 Li and 5 ,mol L,1 KBR the extrasystole was abolished in 77%. Lithium and KBR effects particularly for the extrasystole can be explained through the inhibition of Ca entry via Na,Ca exchange giving support to the participation of the Na,Ca exchanger in the Ca influx from the extracellular space. In addition, the results also suggest the possibility of an effect of Li on an additional Ca sensitive locus (different than the Na,Ca exchanger). In this connection, in isolated contractions lithium decreased the energy release fraction related to mitochondrial processes (H4) increasing the economy of the overall cardiac contraction. [source]

Learning in a geometric model of place cell firing

HIPPOCAMPUS, Issue 9 2007
Caswell Barry
Abstract Following Hartley et al. (Hartley et al. (2000) Hippocampus 10:369,379), we present a simple feed-forward model of place cell (PC) firing predicated on neocortical information regarding the environmental geometry surrounding the animal. Incorporating the idea of boundaries with distinct sensory qualities, we show that synaptic plasticity mediated by a BCM-like rule (Bienenstock et al. (1982) J Neurosci 2:32,48) produces PCs that encode position relative to specific extended landmarks. In an unchanging environment the model is shown to undergo an initial phase of learning, resulting in the formation of stable place fields. In familiar environments, perturbation of environmental cues produces graded changes in the firing rate and position of place fields. Model simulations are compared favorably with three sets of experimental data: (1) Results published by Barry et al. (Barry et al. (2006) Rev Neurosci 17:71,97) showing the slow disappearance of duplicate place fields produced when a barrier is placed into a familiar environment. (2) Rivard et al.'s (Rivard et al. (2004) J Gen Physiol 124:9,25) study showing a graded response in PC firing such that fields near to a centrally placed object encode space relative to the object, whereas more distant fields respond to the surrounding environment. (3) Fenton et al.'s (Fenton et al. (2000a) J Gen Physiol 116:191,209) observation that inconsistent rotation of cue cards produces parametric changes in place field positions. The merits of the model are discussed in terms of its extensibility and biological plausibility. © 2007 Wiley-Liss, Inc. [source]

The origin of fibroblasts and mechanism of cardiac fibrosis

Guido Krenning
Fibroblasts are at the heart of cardiac function and are the principal determinants of cardiac fibrosis. Nevertheless, cardiac fibroblasts remain poorly characterized in molecular terms. Evidence is evolving that the cardiac fibroblast is a highly heterogenic cell population, and that such heterogeneity is caused by the distinct origins of fibroblasts in the heart. Cardiac fibroblasts can derive either from resident fibroblasts, from endothelial cells via an endothelial,mesenchynmal transition or from bone marrow-derived circulating progenitor cells, monocytes and fibrocytes. Here, we review the function and origin of fibroblasts in cardiac fibrosis.NB. The information given is correct. J. Cell. Physiol. 225: 631,637, 2010. © 2010 Wiley-Liss, Inc. [source]

c-Jun N-terminal kinase is largely involved in the regulation of tricellular tight junctions via tricellulin in human pancreatic duct epithelial cells

Takashi Kojima
Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c-Jun N-terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12- O -tetradecanoylphorbol 13-acetate (TPA), and the proinflammatory cytokines IL-1,, TNF,, and IL-1,. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL-1,, TNF,, and IL-1,, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT-transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells. J. Cell. Physiol. 225: 720,733, 2010. © 2010 Wiley-Liss, Inc. [source]

Shedding of microparticles by myofibroblasts as mediator of cellular cross-talk during normal wound healing

Véronique J. Moulin
Interactions between cells are a crucial mechanism to correctly heal a wounded tissue. Myofibroblasts have a central role during healing but their means to communicate with other cells is unknown. Microparticles (MP) have demonstrated a potential role as mediators of cellular interactions during various diseases. We have analyzed the production of MP by normal (Wmyo) and pathological (hypertrophic scar, Hmyo) myofibroblasts and human dermal fibroblasts (Fb) when treated with serum or plasma as examples of body fluids. We have shown that the presence of these body fluids induced a very significant increase in MP production by Wmyo while no MP production was denoted for Hmyo and Fb. These effects were at least due to thermally sensitive protein(s) with a molecular mass >30,kDa. Furthermore, the increase in MP production was not linked to an increase in apoptotic Wmyo. MP characterization showed that VEGF and FGF2 were present in MP and that endothelial and (myo)fibroblast cell growth can be stimulated by MP treatment. We postulated that MP production by myofibroblasts could modulate mesenchymal cell growth and angiogenesis during normal healing. J. Cell. Physiol. 225: 734,740, 2010. © 2010 Wiley-Liss, Inc. [source]

Elevated expression of TMEM205, a hypothetical membrane protein, is associated with cisplatin resistance

Ding-Wu Shen
Development of cisplatin resistance in cancer cells appears to be a consequence of multiple epigenetic alterations in genes involved in DNA damage repair, proto-oncogenes, apoptosis, transporters, transcription factors, etc. In this study, we found that expression of the hypothetical transmembrane protein TMEM205 (previously known as MBC3205) is associated with cisplatin resistance. TMEM205 was first detected by functional cloning from a retroviral cDNA library made from human cisplatin-resistant (CP-r) cells. TMEM205 is predicted to be a transmembrane protein, but its expression, localization, and function have not previously been investigated. A polyclonal antibody directed to the TMEM205 protein was raised in our laboratory. Using this antibody, it was demonstrated that this protein is located at the cell surface. Its expression is increased in our cisplatin-selected CP-r cell lines, as demonstrated by immunoblotting, confocal examination, and immuno-electron microscopy. Stable transfection of the TMEM205 gene confers resistance to cisplatin by approximately 2.5-fold. Uptake assays with Alexa Fluor-cisplatin showed reduced accumulation in CP-r KB-CP.3 and KB-CP.5 cells, and in TMEM205-transfected cells. Analysis of TMEM205 expression profiles in normal human tissues indicates a differential expression pattern with higher expression levels in the liver, pancreas, and adrenal glands. These results indicate that a novel mechanism for cisplatin resistance is mediated by TMEM205, and also suggest that overexpression of TMEM205 in CP-r cells may be valuable as a biomarker or target in cancer chemotherapy. J. Cell. Physiol. 225: 822,828, 2010. © 2010 Wiley-Liss, Inc. [source]

Role of calcium and ROS in cell death induced by polyunsaturated fatty acids in murine thymocytes

Aparna Prasad
We investigated the mechanisms whereby omega-3 and -6 polyunsaturated fatty acids (PUFAs) cause cell death of mouse thymocytes using flow cytometry, focusing on the respective roles of intracellular calcium concentration, [Ca2+]i and reactive oxygen species (ROS). We applied the C-22, 20, and 18 carbon omega-3 (DHA, EPA, ALA) and omega-6 (DTA, ARA, and LNA) fatty acids to isolated thymocytes and monitored cell death using the DNA-binding dye, propidium iodide. When applied at 20,µM concentration, omega-3 fatty acids killed thymocytes over a period of 1,h with a potency of DHA,>,EPA,>,ALA. The omega-6 PUFAs were more potent. The C18 omega-6 fatty acid, LNA, was the most potent, followed by DHA and ARA. Cell death was always accompanied by an increase in the levels of [Ca2+]i and ROS. Both increases were in proportion to the potency of the PUFAs in inducing cell death. Removing extracellular calcium did not prevent the elevation in [Ca2+]i nor cell death. However, the intracellular calcium chelator, BAPTA, almost totally reduced both the elevation in [Ca2+]i and cell death, while vitamin E reduced the elevation in ROS and cell death. BAPTA also prevented the elevation in ROS, but vitamin E did not prevent the elevation in [Ca2+]i. Thapsigargin, which depletes endoplasmic reticulum calcium, blocked the elevation in [Ca2+]i, but CCCP, a mitochondrial calcium uptake inhibitor, did not. These results suggest that the six PUFAs we studied kill thymocytes by causing release of calcium from endoplasmic reticulum, which causes release of ROS from mitochondria which leads to cell death. J. Cell. Physiol. 225: 829,836, 2010. © 2010 Wiley-Liss, Inc. [source]

Origin of cardiac progenitor cells in the developing and postnatal heart

Elizabeth N. Kuhn
The mammalian heart lacks the capacity to replace the large numbers of cardiomyocytes lost due to cardiac injury. Several different cell-based routes to myocardial regeneration have been explored, including transplantation of cardiac progenitors and cardiomyocytes into injured myocardium. As seen with cell-based therapies in other solid organ systems, inherent limitations, such as host immune response, cell death and long-term graft instability have hampered meaningful cardiac regeneration. An understanding of the cell biology of cardiac progenitors, including their developmental origin, lineage markers, renewal pathways, differentiation triggers, microenvironmental niche, and mechanisms of homing and migration to the site of injury, will enable further refinement of therapeutic strategies to enhance clinically meaningful cardiac repair. J. Cell. Physiol. 225: 321,325, 2010. © 2010 Wiley-Liss, Inc. [source]

Syntaxin 16: Unraveling cellular physiology through a ubiquitous SNARE molecule

Yanan Chen
Syntaxin 16 (Syx16) is member of the soluble N -ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of molecules that functions in membrane fusion in eukaryotic cells. A rather ubiquitously expressed, tail-anchored membrane protein localized mainly at the trans-Golgi network (TGN), it mediates primarily retrograde endosomal-TGN transport. In spite of its ubiquitous expression, Syx16 has specific and interesting roles in the physiology of specialized cells, including Glut4 dynamics, dendritic outgrowth-related membrane traffic, and cytokinesis. We discussed these physiological functions of Syx16 in the light of what is known of its subcellular localization, vesicular trafficking pathways involved, cognate SNARE partners and other interacting proteins. Further, we speculate on some possible pathophysiological roles of Syx16. J. Cell. Physiol. 225: 326,332, 2010. © 2010 Wiley-Liss, Inc. [source]

Epigenetic pre-patterning and dynamics during initial stages of mammalian preimplantation development

Theodore P. Rasmussen
Mammals, like all multicellular organisms, develop from a single cell,the totipotent zygote. During preimplantation development and subsequent development in utero, over 200 distinct cell types are established and integrated into the organ systems and tissues of the developing organism. Much of the field of mammalian developmental biology is devoted to investigation of mechanisms that govern the formation of complete organs and tissues. In contrast to later development, which consumes the vast majority of time associated with development in utero, preimplantation development and germ layer specification occur rapidly. Yet knowledge is limited regarding the regulatory mechanisms that specify the transient, but pluripotent, cellular lineages that form during the initial stages of mammalian development. Gametogenesis and preimplantation development are marked by dramatic and pervasive epigenetic changes rooted in chromatin dynamics. The fundamental mechanisms that specify subsequent cellular lineages of the conceptus are only now becoming understood, and tend to rely relatively heavily upon broad epigenetic mechanisms in addition to master transcription factors. This review considers epigenetic regulation in the very earliest stages of preimplantation development. In addition, recent advances which indicate that some epigenetic coding is imposed during gametogenesis and maintained during preimplantation development are considered. J. Cell. Physiol. 225: 333,336, 2010. © 2010 Wiley-Liss, Inc. [source]

Restoration of DNA-binding and growth-suppressive activity of mutant forms of p53 via a PCAF-mediated acetylation pathway,

Ricardo E. Perez
Tumor-derived mutant forms of p53 compromise its DNA binding, transcriptional, and growth regulatory activity in a manner that is dependent upon the cell-type and the type of mutation. Given the high frequency of p53 mutations in human tumors, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy. In support of this possibility p53 mutants possess a certain degree of conformational flexibility that allows for re-induction of function by a number of structurally different artificial compounds or by short peptides. This raises the question of whether physiological pathways for p53 mutant reactivation also exist and can be exploited therapeutically. The activity of wild-type p53 is modulated by various acetyl-transferases and deacetylases, but whether acetylation influences signaling by p53 mutant is still unknown. Here, we show that the PCAF acetyl-transferase is down-regulated in tumors harboring p53 mutants, where its re-expression leads to p53 acetylation and to cell death. Furthermore, acetylation restores the DNA-binding ability of p53 mutants in vitro and expression of PCAF, or treatment with deacetylase inhibitors, promotes their binding to p53-regulated promoters and transcriptional activity in vivo. These data suggest that PCAF-mediated acetylation rescues activity of at least a set of p53 mutations. Therefore, we propose that dis-regulation of PCAF activity is a pre-requisite for p53 mutant loss of function and for the oncogenic potential acquired by neoplastic cells expressing these proteins. Our findings offer a new rationale for therapeutic targeting of PCAF activity in tumors harboring oncogenic versions of p53. J. Cell. Physiol. 225: 394,405, 2010. © 2010 Wiley-Liss, Inc. [source]

Regulation of lipopolysaccharide-induced inflammatory response and endotoxemia by ,-arrestins,

Katie J. Porter
,-Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that ,-arrestin-1 (,-arr-1) and -2 knockout (KO) mice are protected from TLR4-mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild-type (WT) and ,-arr-1 and -2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS-induced inflammatory cytokine levels in the plasma were markedly decreased in both ,-arr-1 and -2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11b+ and CD11b, populations) from WT, ,-arr-1, and -2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS-induced inflammatory cytokines were significantly blocked in both splenocyte populations from the ,-arr-2 KO compared to the WT mice. This effect in the ,-arr-1 KO mice, however, was restricted to the CD11b, splenocytes. Our studies further indicate that regulation of cytokine production by ,-arrestins is likely independent of MAPK and I,B,-NF,B pathways. Our results, however, suggest that LPS-induced chromatin modification is dependent on ,-arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by ,-arrestins in vivo. Taken together, these results indicate that ,-arr-1 and -2 mediate LPS-induced cytokine secretion in a cell-type specific manner and that both ,-arrestins have overlapping but non-redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406,416, 2010. © 2010 Wiley-Liss, Inc. [source]

CXCR4-independent rescue of the myeloproliferative defect of the gata1low myelofibrosis mouse model by Aplidin®,

Maria Verrucci
The discovery of JAK2 mutations in Philadelphia-negative myeloproliferative neoplasms has prompted investigators to evaluate mutation-targeted treatments to restore hematopoietic cell functions in these diseases. However, the results of the first clinical trials with JAK2 inhibitors are not as promising as expected, prompting a search for additional drugable targets to treat these disorders. In this paper, we used the hypomorphic Gata1low mouse model of primary myelofibrosis (PMF), the most severe of these neoplasms, to test the hypothesis that defective marrow hemopoiesis and development of extramedullary hematopoiesis in myelofibrosis is due to insufficient p27Kip1 activity and is treatable by Aplidin®, a cyclic depsipeptide that activates p27Kip1 in several cancer cells. Aplidin® restored expression of Gata1 and p27Kip1 in Gata1low hematopoietic cells, proliferation of marrow progenitor cells in vitro and maturation of megakaryocytes in vivo (reducing TGF-,/VEGF levels released in the microenvironment by immature Gata1low megakaryocytes). Microvessel density, fibrosis, bone growth, and marrow cellularity were normal in Aplidin®-treated mice and extramedullary hematopoiesis did not develop in liver although CXCR4 expression in Gata1low progenitor cells remained low. These results indicate that Aplidin® effectively alters the natural history of myelofibrosis in Gata1low mice and suggest this drug as candidate for clinical evaluation in PMF. J. Cell. Physiol. 225: 490,499, 2010. © 2010 Wiley-Liss, Inc. [source]

Evidences of a role for eukaryotic translation initiation factor 5A (eIF5A) in mouse embryogenesis and cell differentiation

Lucas T. Parreiras-e-Silva
Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation. J. Cell. Physiol. 225: 500,505, 2010. © 2010 Wiley-Liss, Inc. [source]

Expression of GFAT1 and OGT in podocytes: Transport of glucosamine and the implications for glucose uptake into these cells

Dorota Rogacka
Glutamine:fructose-6-phosphate amidotransferase (GFAT) and N -acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O -glycosylation, one of key post-translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT-PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3,±,0.2,nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1,±,0.2,nmol/min/mg protein) in a significant part, involved a sodium-dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50,µM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6,mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment. J. Cell. Physiol. 225: 577,584, 2010. © 2010 Wiley-Liss, Inc. [source]

Presence of a functional receptor for GLP-1 in osteoblastic cells, independent of the cAMP-linked GLP-1 receptor

Bernardo Nuche-Berenguer
Glucagon-like peptide 1 (GLP-1) controls glucose metabolism in extrapancreatic tissues through receptors other than the pancreatic cAMP-linked GLP-1 receptor; also, GLP-1 induces an insulin- and PTH-independent bone anabolic action in insulin-resistant and type-2 diabetic rats. Here we searched for the presence and characteristics of GLP-1 receptors in osteoblastic MC3T3-E1 cells. [125I]-GLP-1 specific binding to MC3T3-E1 cells was time- and temperature-dependent, reaching maximal value at 30,min at 25°C; in these conditions, [125I]-GLP-1 binding was dissociable, and displaced by GLP-1, partially by GLP-2, but not by exendin-4 (Ex-4), exendin-9 (Ex-9), glucagon or insulin; Scatchard analysis of the unlabeled GLP-1 data showed high and low affinity binding sites; cross-linking of GLP-1 binding revealed an estimated 70,kDa band, almost undetectable in the presence of 10,6,M GLP-1. GLP-1, Ex-9, insulin or glucagon failed to modify cellular cAMP content, while GLP-2 and Ex-4 increased it. However, GLP-1 induced an immediate hydrolysis of glycosylphosphatidylinositols (GPIs) generating short-lived inositolphosphoglycans (IPGs), and an increase in phosphatidylinositol-3 kinase (PI3K) and mitogen activated protein kinase (MAPK) activities; Ex-4 also affected GPIs, but its action was delayed with respect to that of GLP-1. This incretin was found to decrease Runx2 but increased osteocalcin gene expression, without affecting that of osteoprotegerin or the canonical Wnt pathway activity in MC3T3-E1 cells which do not express the pancreatic GLP-1 receptor. Our data demonstrate for the first time that GLP-1 can directly and functionally interact with osteoblastic cells, possibly through a GPI/IPG-coupled receptor. J. Cell. Physiol. 225: 585,592, 2010. © 2010 Wiley-Liss, Inc. [source]

Advanced oxidation protein products inhibit differentiation and activate inflammation in 3T3-L1 preadipocytes,

Qiu Gen Zhou
Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndromes, a condition with impaired preadipocytes differentiation. In the present study, we tested the hypothesis that AOPPs disturb preadipocyte differentiation. Exposure of 3T3-L1 preadipocytes to increased levels of AOPPs inhibited accumulation of intracellular triglyceride and decreased the expression of the essential markers of matured adipocytes, such as adipocyte fatty-acid-binding protein (aP2), CAAT/enhancer-binding protein (C/EBP)-,, and peroxisome proliferator-activated receptor (PPAR)-,, in response to standard adipogenic induction. Inhibitory effects of AOPPs on preadipocytes differentiation was time sensitive, which occurred at the early stage of differentiation. In the presence of AOPPs, induction of preadipocytes differentiation resulted in upregulated expression of C/EBP homologous protein (CHOP) and CUG-Triplet repeat-binding protein (CUGBP), two important inhibitors of preadipocytes differentiation. In addition, treatment with AOPPs increased abundance of C/EBP-,-liver enriched inhibitory protein (C/EBP-,-LIP), a truncated C/EBP-, isoform without adipogenic activity. Moreover, AOPPs-treated preadipocytes expressed a macrophage marker F4/80 and overexpressed tumor necrosis factor-, and interleukin-6 via nuclear factor-,B (NF-,B)-dependent pathway. However, blocking inflammation with NF-,B inhibitor failed to improve AOPPs-induced inhibition of preadipocytes differentiation. These data suggest that accumulation of AOPPs may inhibit differentiation of preadipocytes and activate inflammation in these cells. This information might have implication for understanding the impairment of preadipocytes differentiation and fat inflammation seen in metabolic syndrome. J. Cell. Physiol. 225: 42,51, 2010. © 2010 Wiley-Liss, Inc. [source]

Reactive oxygen species control senescence-associated matrix metalloproteinase-1 through c-Jun-N-terminal kinase,

Jaya Dasgupta
The lifetime exposure of organisms to oxidative stress influences many aging processes which involve the turnover of the extracellular matrix. In this study, we identify the redox-responsive molecular signals that drive senescence-associated (SA) matrix metalloproteinase-1 (MMP-1) expression. Precise biochemical monitoring revealed that senescent fibroblasts increase steady-state (H2O2) 3.5-fold (13.7,48.6,pM) relative to young cells. Restricting H2O2 production through low O2 exposure or by antioxidant treatments prevented SA increases in MMP-1 expression. The H2O2 -dependent control of SA MMP-1 is attributed to sustained JNK activation and c-jun recruitment to the MMP-1 promoter. SA JNK activation corresponds to increases and decreases in the levels of its activating kinase (MKK-4) and inhibitory phosphatase (MKP-1), respectively. Enforced MKP-1 expression negates SA increases in JNK phosphorylation and MMP-1 production. Overall, these studies define redox-sensitive signaling networks regulating SA MMP-1 expression and link the free radical theory of aging to initiation of aberrant matrix turnover. J. Cell. Physiol. 225: 52,62, 2010. © 2010 Wiley-Liss, Inc. [source]

Role of TNF alpha and PLF in bone remodeling in a rat model of repetitive reaching and grasping,

Shobha Rani
We have previously developed a voluntary rat model of highly repetitive reaching that provides an opportunity to study effects of non-weight bearing muscular loads on bone and mechanisms of naturally occurring inflammation on upper limb tissues in vivo. In this study, we investigated the relationship between inflammatory cytokines and matricellular proteins (Periostin-like-factor, PLF, and connective tissue growth factor, CTGF) using our model. We also examined the relationship between inflammatory cytokines, PLF and bone formation processes. Rats underwent initial training for 5 weeks, and then performed a high repetition high force (HRHF) task (12,reaches/min, 60% maximum grip force, 2,h/day, 3 days/week) for 6 weeks. We then examined the effect of training or task performance with or without treatment with a rat specific TNF, antibody on inflammatory cytokines, osteocalcin (a bone formation marker), PLF, CTGF, and behavioral indicators of pain or discomfort. The HRHF task decreased grip strength and induced forepaw mechanical hypersensitivity in both trained control and 6-week HRHF animals. Two weeks of anti-TNF, treatment improved grip strength in both groups, but did not ameliorate forepaw hypersensitivity. Moreover, anti-TNF, treatment attenuated task-induced increases in inflammatory cytokines (TNF,, IL-1,, and MIP2 in serum; TNF, in forelimb bone and muscles) and serum osteocalcin in 6-week HRHF animals. PLF levels in forelimb bones and flexor digitorum muscles increased significantly in 6-week HRHF animals, increases attenuated by anti-TNF, treatment. CTGF levels were unaffected by task performance or anti-TNF, treatment in 6-week HRHF muscles. In primary osteoblast cultures, TNF,, MIP2 and MIP3a treatment increased PLF levels in a dose dependent manner. Also in primary osteoblast cultures, increased PLF promoted proliferation and differentiation, the latter assessed by measuring Runx2, alkaline phosphatase (ALP) and osteocalcin mRNA levels; ALP activity; as well as calcium deposition and mineralization. Increased PLF also promoted cell adhesion in MC3T3-E1 osteoblast-like cell cultures. Thus, tissue loading in vivo resulted in increased TNF,, which increased PLF, which then induced anabolic bone formation, the latter results confirmed in vitro. J. Cell. Physiol. 225: 152,167, 2010. © 2010 Wiley-Liss, Inc. [source]

Angiotensin II/angiotensin II type I receptor (AT1R) signaling promotes MCF-7 breast cancer cells survival via PI3-kinase/Akt pathway

Yanbin Zhao
Angiotensin II (Ang II) is a bioactive peptide of the renin,angiotensin system, acting not only as a vasoconstrictor but also as a growth promoter via angiotensin II type 1 receptor (AT1R) in some cancer. In this study, we examined the mechanisms by which Ang II affected the cell proliferation in AT1R-positive MCF-7 human breast cancer cells. Ang II stimulated the growth of breast cancer cells in a dose- and time-dependent manner. The maximal proliferation effect on MCF-7 cells was obtained with 10,4,M Ang II at 24,h. Losartan (10,5,M, an AT1R antagonist) significantly decreased the level of Ang-II-induced proliferative effects, whereas PD123319 (10,5,M, an AT2R antagonist) had no effects. Moreover, Ang II could significantly accelerate S-phase progression, which was inhibited by losartan (10,5,M) or LY294002 (50,µM, a PI3-kinase inhibitor). In addition, Ang II caused rapid activation of p-Akt in a dose- and time-dependent manner. 10,4,M Ang II induced a significant increase of p-Akt in 15,min. The peak level of p-Akt could be persisted for at least 6,h. Among the signaling molecules downstream of Akt, we revealed that Ang II also significantly upregulated CyclinD1, GSK3,, and downregulated p27. Pretreatment with losartan (10,5,M) or LY294002 (50,µM) could significantly suppress these effects of Ang II. These findings suggest that Ang II plays a role in the growth of AT1R-positive breast cancer cells through PI3-kinase/Akt pathway activation. Therefore, targeting Ang II/AT1R signaling could be a novel therapeutic for breast cancer. J. Cell. Physiol. 225: 168,173, 2010. © 2010 Wiley-Liss, Inc. [source]

B-type natriuretic peptide and extracellular matrix protein interactions in human cardiac fibroblasts

Brenda K. Huntley
Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix (ECM) proteins. B-type natriuretic peptide (BNP) is anti-fibrotic, inhibits collagen production, augments matrix metalloproteinases, and suppresses CF proliferation. Recently, we demonstrated that the ECM protein fibronectin (FN) augmented production of BNP's second messenger, 3,, 5, cyclic guanosine monophosphate (cGMP) in CFs, supporting crosstalk between FN, BNP, and its receptor, natriuretic peptide receptor A (NPR-A). Here, we address the specificity of FN to augment cGMP generation by investigating other matrix proteins, including collagen IV which contains RGD motifs and collagen I and poly- L -lysine, which have no RGD domain. Collagen IV showed increased cGMP generation to BNP similar to FN. Collagen I and poly- L -lysine had no effect. As FN also interacts with integrins, we then examined the effect of integrin receptor antibody blockade on BNP-mediated cGMP production. On FN plates, antibodies blocking RGD-binding domains of several integrin subtypes had little effect, while a non-RGD domain interfering integrin ,v,3 antibody augmented cGMP production. Further, on uncoated plates, integrin ,v,3 blockade continued to potentiate the BNP/cGMP response. These studies suggest that both RGD containing ECM proteins and integrins may interact with BNP/NPR-A to modulate cGMP generation. J. Cell. Physiol. 225: 251,255, 2010. © 2010 Wiley-Liss, Inc. [source]

Shed HER2 extracellular domain in HER2-mediated tumor growth and in trastuzumab susceptibility,

Gaia C. Ghedini
The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti-HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2-driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110-kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, -2, and -3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2-driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2-driven SKOV3 tumor growth but not that of HER2-negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD-facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2-disease and in the susceptibility to Trastuzumab-based therapy. J. Cell. Physiol. 225: 256,265, 2010. © 2010 Wiley-Liss, Inc. [source]

Cross-talk between the insulin-like growth factor (IGF) axis and membrane integrins to regulate cell physiology

James Beattie
The biology of cross-talk between activated growth factor receptors and cell-surface integrins is an area which has attracted much interest in recent years (Schwartz and Ginsberg, 2002). This review discusses the relationship between the insulin-like growth factor (IGF) axis and cell-surface integrin receptors in the regulation of various aspects of cell physiology. Key to these interactions are signals transmitted between integrins and the IGF-I receptor (IGF-IR) when either or both are bound to their cognate ligands and we will review the current state of knowledge in this area. The IGF axis comprises many molecular components and we will also discuss the potential role of these species in cross-talk with the integrin receptor. With respect to integrin ligands, we will mainly focus on the well-characterized interactions of the two extracellular matrix (ECM) glycoproteins fibronectin (FN) and vitronectin (VN) with cell-surface ligands, and, how this affects activity through the IGF axis. However, we will also highlight the importance of other integrin activation mechanisms and their impact on IGF activity. J. Cell. Physiol. 224: 605,611, 2010. © 2010 Wiley-Liss, Inc. [source]

Thyroid tumor marker genomics and proteomics: Diagnostic and clinical implications

Angelo Carpi
Two systems biology concepts, genomics and proteomics, are highlighted in this review. These techniques are implemented to optimize the use of thyroid tumor markers (TTM). Tissue microarray studies can produce genetic maps and proteomics, patterns of protein expression of TTM derived from preoperative biopsies and specimens. For instance, papillary and medullary thyroid cancers harbor RAS, RET, and BRAF genetic mutations. Follicular thyroid cancers harbor translocations and fusions of certain genes (PAX 8 and PPAR-gamma). Proteomic analysis from various tissue sources can provide useful information regarding the overall state of a thyroid cancer cell. Understanding the molecular events related to these genetic and protein alterations can potentially clarify thyroid cancer pathogenesis and guide appropriate molecular targeted therapies. However, despite the realization that these emerging technologies hold great promise, there are still significant obstacles to the routine use of TTM. These include equivocal thyroid nodule tissue morphologic interpretations, inadequate standardization of methods, and monetary costs. Interpretative shortcomings are frequently due to the relative scarcity of cellular material from fine-needle aspiration biopsy (FNAB) specimens. This can be rectified with large needle aspiration biopsy (LNAB) techniques and is exemplified by the favorable performance of galectin-3 determinations on LNAB specimens. J. Cell. Physiol. 224: 612,619, 2010. © 2010 Wiley-Liss, Inc. [source]

Src is a major signaling component for CTGF induction by TGF-,1 in osteoblasts,

X. Zhang
Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor ,1 (TGF-,1) where it acts as a downstream mediator of TGF-,1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk, and Smad signaling for CTGF induction by TGF-,1 in osteoblasts; however, the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-,1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-,1. Additionally, inhibiting Src activation prevented Erk activation, Smads 2 and 3 activation and nuclear translocation by TGF-,1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway directly by mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF-,1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. These data demonstrate that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-,1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. J. Cell. Physiol. 224: 691,701, 2010. © 2010 Wiley-Liss, Inc. [source]

Interferon regulatory factor-1 acts as a powerful adjuvant in tat DNA based vaccination,

Arianna Castaldello
Genetic vaccines are safe cost-effective approaches to immunization but DNA immunization is an inefficient process. There is, therefore, a pressing need for adjuvants capable of enhancing the immunogenicity and effectiveness of these vaccines. This is particularly important for diseases for which successful vaccines are still lacking, such as cancer and infectious diseases including HIV-1/AIDS. Here we report an approach to enhance the immunogenicity of DNA vaccines involving the use of transcription factors of the Interferon regulatory factor (IRF) family, specifically IRF-1, IRF-3, and IRF-7 using the tat gene as model antigen. Balb/c mice were immunized by three intramuscular inoculations, using a DNA prime-protein boost protocol, with a DNA encoding tat of HIV-1 and the indicated IRFs and immune responses were compared to those induced by vaccination with tat DNA alone. In vivo administration of plasmid DNA encoding IRF-1, or a mutated version of IRF-1 deleted of the DNA-binding domain, enhanced Tat-specific immune responses and shifted them towards a predominant T helper 1-type immune response with increased IFN-, production and cytotoxic T lymphocytes responses. Conversely, the use of IRF-3 or IRF-7 did not affect the tat -induced responses. These findings define IRF-1 and its mutated form as efficacious T helper 1-inducing adjuvants in the context of tat- based vaccination and also providing a new promising candidate for genetic vaccine development. J. Cell. Physiol. 224: 702,709, 2010. © 2010 Wiley-Liss, Inc. [source]

Local regulation of human breast xenograft models,,

Jodie M. Fleming
Breast cancer studies implant human cancer cells under the renal capsule, subcutaneously, or orthotopically and often use estrogen supplementation and immune suppressants (etoposide) in xenograft mouse models. However, cell behavior is significantly impacted by signals from the local microenvironment. Therefore, we investigated how the combinatorial effect of the location of injection and procedural differences affected xenograft characteristics. Patient-derived breast cancer cells were injected into mouse abdominal or thoracic mammary glands,±,estrogen and/or etoposide pretreatment. Abdominal xenografts had increased tumor incidence and volume, and decreased latency (P,<,0.001) compared to thoracic tumors. No statistically significant difference in tumor volume was found in abdominal xenografts treated,±,estrogen or etoposide; however, etoposide suppressed tumor volume in thoracic xenografts (P,<,0.02). The combination of estrogen and etoposide significantly decreased tumor incidence in both sites. In addition, mice treated,±,estradiol were injected orthotopically or subcutaneously with well-characterized breast cancer cell lines (MCF7, ZR75-1, MDA MB-231, or MCF10Ca1h). Orthotopic injection increased tumor volume; growth varied with estrogen supplementation. Location also altered methylation status of several breast cancer-related gene promoters. Lastly, vascularization of orthotopic tumors was significantly enhanced compared to subcutaneous tumors. These data suggest that optimal xenograft success occurs with orthotopic abdominal injections and illustrate molecular details of the compelling influence of the local microenvironment on in vivo models. J. Cell. Physiol. 224: 795,806, 2010. Published 2010 Wiley-Liss, Inc. [source]

Ex vivo organ culture of adipose tissue for in situ mobilization of adipose-derived stem cells and defining the stem cell niche

Young-Il Yang
In spite of the advances in the knowledge of adipose-derived stem cells (ASCs), in situ location of ASCs and the niche component of adipose tissue (AT) remain controversial due to the lack of an appropriate culture system. Here we describe a fibrin matrix-supported three-dimensional (3D) organ culture system for AT which sustains the ASC niche and allows for in situ mobilization and expansion of ASCs in vitro. AT fragments were completely encapsulated within the fibrin matrix and cultured under dynamic condition. The use of organ culture of AT resulted in a robust outgrowth and proliferation in the fibrin matrix. The outgrown cells were successfully recovered from fibrin by urokinase treatment. These outgrown cells fulfilled the criteria of mesenchymal stem cells, adherence to plastic, multilineage differentiation, and cell surface molecule expression. In vitro label retaining assay revealed that newly divided cells during the culture resided in interstitium between adipocytes and capillary endothelial cells. These interstitial stromal cells proliferated and outgrew into the fibrin matrix. Both in situ mobilized and outgrown cells expressed CD146 and ,-smooth muscle actin (SMA), but no endothelial cell markers (CD31 and CD34). The structural integrity and spatial approximation of CD31,/CD34,/CD146+/SMA+ interstitial stromal cells, adipocytes, and capillary endothelial cells were well preserved during in vitro culture. Our results suggest that ASCs are natively associated with the capillary wall and more specifically, belong to a subset of pericytes. Furthermore, organ culture of AT within a fibrin matrix-supported 3D environment can recapitulate the ASC niche in vitro. J. Cell. Physiol. 224: 807,816, 2010. © 2010 Wiley-Liss, Inc. [source]

Shepherding AKT and androgen receptor by Ack1 tyrosine kinase

Kiran Mahajan
Ack1 (also known as ACK, TNK2, or activated Cdc42 kinase) is a structurally unique non-receptor tyrosine kinase that is expressed in diverse cell types. It integrates signals from plethora of ligand-activated receptor tyrosine kinases (RTKs), for example, MERTK, EGFR, HER2, PDGFR and insulin receptor to initiate intracellular signaling cascades. Ack1 transduces extracellular signals to cytosolic and nuclear effectors such as the protein kinase AKT/PKB and androgen receptor (AR), to promote cell survival and growth. While tyrosine phosphorylation of AR at Tyr267 regulates androgen-independent recruitment of AR to the androgen-responsive enhancers and transcription of AR target genes to drive prostate cancer progression, phosphorylation of an evolutionarily conserved Tyrosine 176 in the kinase domain of AKT is essential for mitotic progression and positively correlates with breast cancer progression. In contrast to AR and AKT, Ack1-mediated phosphorylation of the tumor suppressor Wwox at Tyr287 lead to rapid Wwox polyubiquitination followed by degradation. Thus, by its ability to promote tumor growth by negatively regulating tumor suppressor such as Wwox and positively regulating pro-survival factors such as AKT and AR, Ack1 is emerging as a critical player in cancer biology. In this review, we discuss recent advances in understanding the physiological functions of Ack1 signaling in normal cells and the consequences of its hyperactivation in various cancers. J. Cell. Physiol. 224: 327,333, 2010. © 2010 Wiley-Liss, Inc. [source]