AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background: Activated hepatic stellate cells (HSCs) are closely involved in the initiation, perpetuation, and resolution of liver fibrosis. Pro-inflammatory cytokine levels are positively correlated with the transition from liver injury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis. Methods: In this study, we investigated the effect of the pro-inflammatory cytokine interleukin (IL)-1β on the proliferation and signaling pathways involved in fibrogenesis in LX-2 cells, an HSC cell line, using western blotting and cell proliferation assays. Results: IL-1β increased the proliferation rate and α-smooth muscle actin (SMA) expression of LX-2 cells in a dose-dependent manner. Within 1 h after IL-1β treatment, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-κB (NF-κB) signaling was activated in LX-2 cells. Subsequently, protein kinase B (AKT) phosphorylation and an increase in α- SMA expression were observed in LX-2 cells. Each inhibitor of JNK, p38, or NF-κB decreased cell proliferation, AKT phosphorylation, and α-SMA expression in IL-1β-treated LX-2 cells. Conclusion: These results indicate that JNK, p38, and NF-κB signals converge at AKT phosphorylation, leading to LX-2 activation by IL-1β. Therefore, the AKT signaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.     

Mechanical vibrations increase the proliferation of articular chondrocytes in high-density culture

Tissue engineering is a promising approach for articular cartilage repair; however, it still has proven a challenge to produce tissue from the limited number of cells that can be extracted from a single individual. Relatively few cell expansion methods exist without the problems of dedifferentiation and/or loss of potency. Previously, it has been shown that mechanical vibrations can enhance chondrocyte proliferation in monolayer culture. Thus, it was hypothesized that chondrocytes grown in high-density culture would respond in a similar fashion while maintaining phenotypic stability. Isolated bovine articular chondrocytes were seeded in high-density culture on Millicell filters and subjected to mechanical vibrations 48 h after seeding. Mechanical vibrations enhanced chondrocyte proliferation at frequencies above 350 Hz, with the peak response occurring at a 1g amplitude for a duration of 30 min. Under these conditions, the gene expression of cartilage-specific and dedifferentiation markers (collagen II, collagen I, and aggrecan) were unchanged by the imposed stimulus. To determine the effect of accumulated extracellular matrix (ECM) on this proliferative response, selected cultures were stimulated under the same conditions after varying lengths of preculture. The amount of accumulated ECM (collagen and proteoglycans) decreased this proliferative response, with the cultures becoming insensitive to the stimulus after 1 week of preculture. Thus, mechanical vibration can serve as an effective means preferentially to stimulate the proliferation of chondrocytes during culture, but its effects appear to be limited to the early stages where ECM accumulation is at a minimum.     

Apigenin inhibits cell migration through MAPK pathways in human bladder smooth muscle cells

Apigenin, a nonmutagenic flavonoid, has been shown to possess free radical scavenging activities, anticarcinogenic properties, antioxidant and anti-inflammatory effects. Recently, apigenin was reported to cause gastric relaxation in murine. To assess possible effects of apigenin on migration of bladder smooth muscle (SM) cell, we isolated SM cells from peri-cancer tissue of human bladder and established a cell model that was capable to overexpress transiently MEKK1 (MEK kinase 1). Results showed that overexpression of active human MEKK1 by adenoviruses infection induced migration of human bladder smooth muscle (hBSM) cells and phosphorylation of MAPKs, ERK, JNK and p38, which are the downstream molecules of MEKK1. Then, hBSM cell overexpressing MEKK1 were exposed to apigenin (50 microM). Our data indicated that apigenin inhibited significantly activation/phosphorylation of MAPKs and migration of hBSM cells induced by MEKK1 overexpression. Besides, apigenin inhibited actin polymerization, which underlines muscle contraction and cell migration. The results suggest that apigenin inhibits activation of MAPKs and thereby the cell migration. The mechanism might be that apigenin blocks signal transmission from MEKK1 to MAPKs.     

Mechanosensory neurons, cutaneous mechanoreceptors, and putative mechanoproteins

The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid-sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock-out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited.     

Cell and matrix morpho-functional analysis in chondrocyte micromasses

Micromass cultures represent a convenient means of studying chondrocyte physiology in the context of a tridimensional culture model. In this study, we present the first ultrastructural analysis of the distribution and organization of the extracellular components in micromasses in comparison with their cartilaginous counterparts. Primary chondrocytes obtained from osteoarthritis patients were pelleted in micromasses. Transmission electron microscopy and immunofluorescence were used to evaluate the distribution of major extracellular matrix proteins, i.e., aggrecan, chondroitin-4-sulfate, chondroitin-6-sulfate, and collagen I and II. Both approaches revealed a number of morphological features shared by micromass and cartilage chondrocytes. In particular, in micromasses, chondrocytes are in close contact with an organized extracellular matrix that adequately mimics that of cartilage. Cells were observed to establish specialized junctions for cell-extracellular matrix crosstalk. Noteworthy, cells seem endowed in a chondroitin sulfate-rich microenvironment, and thus possibly ensuring the immobilization of chemokines, a family of molecules emerging in osteoarthritis pathogenesis, in a haptotactic-like gradient to the chondrocytes, which facilitates the binding to their receptors. To determine the suitability of this model to investigate osteoarthritis pathogenesis, a potential apoptotic stimulus (endothelial IL-8) was used, and ultrastructural analysis assessed apoptosis induction. Micromass cultures were proved to be an experimental technique providing a large number of properly differentiated chondrocytes, and thus allowing reliable biochemical and morphological studies. They represent, therefore, a novel approach to osteoarthritis investigation that promises more thorough understanding of chondrocyte physiology in osteoarthritis.     

Light-controlled phosphorylation in the TrkA-Y785 site by photosensitive UAAs activates the MAPK/ERK signaling pathway

Background: This paper aims to establish a light-controlled phosphorylation detection method at the Y785 site of tropomyosin receptor kinase A (TrkA) receptor in mammalian cells by using genetic code expansion technology and detecting the effects of optical activation of this site on the downstream MAPK/ERK pathway. The study is based on the current situation that the regulatory mechanism of TrkA phosphorylation has not been fully elucidated. Methods: Two photosensitive unnatural amino acids, p-azido-L-phenylalanine (AzF) and photo-caged tyrosine (ONB) were introduced into the TrkA-Y785 site by genetic code expansion technology and site-directed mutagenesis. Western blotting and laser confocal imaging were conducted to analyze the effects of this site on activating the MAPK/ERK pathway and nerve cell differentiation before and after photostimulation. Results: Our results supplemented the light-controlled results of the TrkA-Y785 site based on our previous research and verified that Y785 also makes important contributions in regulating the MAPK/ERK pathway. Conclusion: This study demonstrated the significant contributions of the TrkAY785 site in regulating the ERK pathway by precisely controlling the phosphorylation state of a single tyrosine site.     

Deconstructing the molecular mechanisms of cell cycle control in a mouse adrenocortical cell line: roles of ACTH

This is a progress report of an attempt to deconstruct the signaling network underlying cell cycle control in the mouse Y1 adrenocortical cell line, aiming to uncover ACTH growth regulatory pathways. Y1 adrenocortical tumor cells possess amplified and overexpressed c-Ki-ras proto-oncogene. Despite this oncogenic lesion, Y1 cells retain tight regulatory mechanisms of cell cycle control typified by the sequential events comprising the mitogenic response triggered by FGF2 in G0/G1-arrested Y1 cells: 1) activation of ERK1/2 and PI3K, by 5 minutes; 2) induction of c-Fos and c-Myc proteins by 2 hours; 3) induction of cyclin D1 protein by 5 hours; 4) phosphorylation of Rb protein between 6 and 8 hours; 5) onset of DNA synthesis by 8-9 hours. In this cell line, ACTH-receptor (ACTH-R) activates contradictory pathways of growth regulation. First, ACTH coordinately induces fos and jun gene families via activation of both ERK1/2 and cAMP/PKA pathways, resembling a mitogen. Second, ACTH-R triggers cAMP/PKA-mediated antimitogenic mechanisms comprised of Akt/PKB dephosphorylation/deactivation, c-Myc protein degradation, and p27(Kip1) protein induction. Induction of cyclin D1 depends on activation of both ERK1/2 and PI3K, but is not affected by ACTH action. As a consequence, ACTH antagonizes FGF2 mitogenic activity but ectopic expression of the c-Myc protein (via MycER fusion protein) is sufficient to abrogate this ACTH antagonistic effect over FGF2 mitogenic activity. Ectopic expression of both c-Myc and cyclin D1 is not sufficient to drive G0/G1-arrested Y1 cells into S phase, but when the sustained expression of these two proteins is complemented by ACTH treatment it promotes G1 phase progression and DNA synthesis initiation. In conclusion, ACTH-receptor lacks signaling potential sufficient to initiate a mitogenic response in Y1 adrenocortical cells and, therefore, cannot substitute for bona fide mitogens like FGF2.     

An investigation into the effects of the hierarchical structure of tendon fascicles on micromechanical properties

During physiological loading, a tendon is subjected to tensile strains in the region of up to 6 per cent. These strains are reportedly transmitted to cells, potentially initiating specific mechanotransduction pathways. The present study examines the local strain fields within tendon fascicles subjected to tensile strain in order to determine the mechanisms responsible for fascicle extension. A hierarchical approach to the analysis was adopted, involving micro and macro examination. Micro examination was carried out using a custom-designed rig, to enable the analysis of local tissue strains in isolated fascicles, using the cell nuclei as strain markers. In macro examination, a video camera was used to record images of the fascicles during mechanical testing, highlighting the point of crimp straightening and macro failure. Results revealed that local tensile strains within a collagen fibre were consistently smaller than the applied strain and showed no further increase once fibres were aligned. By contrast, between-group displacements, a measure of fibre sliding, continued to increase beyond crimp straightening, reaching a mean value of 3.9 per cent of the applied displacement at 8 per cent strain. Macro analysis displayed crimp straightening at a mean load of 1 N and sample failure occurred through the slow unravelling of the collagen fibres. Fibre sliding appears to provide the major mechanism enabling tendon fascicle extension within the rat-tail tendon. This process will necessarily affect local and cellular strains and consequently mechanotransduction pathways.     

Mechanotransduction-The relationship between gravity,cells and tensile loading in extracellular matrix

Gravity plays a central role in vertebrate development and evolution. Mechanotransduction involves the tensile tethering of veins and arteries, connections between the epidermis and dermis in skin, tensile stress concentrations that occur at tissue interfaces, cell-cell interactions, cell-collagen fiber stress transfer in extracellular matrix and fluid shear flow. While attention in the past has been directed at understanding the myriad of biochemical players associated with mechanotransduction pathways, less attention has been focused on determining the tensile mechanical behavior of tissues in vivo. Fibroblasts sit on the surface of collagen fibers in living skin and exert a retractile force on the fibers. This retractile force pulls against the tension in collagen fibers in skin. After fibroblast-collagen fiber interactions are altered either by changes in fibroblast adhesion or after formation of cancer associated fibroblasts, and changes in cell junctions, alterations in the retractive force leads to changes in mechanotransduction. The purpose of this paper is to present a model of tensile forces that occur at the fibroblast-collagen fiber interface and how these forces are important in extracellular matrix physiology in health and disease.     

Defining mitogen-activated protein kinase pathways with mass spectrometry-based approaches

Mitogen-activated protein kinases are a group of ubiquitously expressed kinase pathways that have been conserved from yeast through humans. They control a large number of critical cell functions. Identification of targets of those kinases is necessary to define signal transduction pathways that lead to cell responses. The application of a number of mass spectrometry-based techniques to the identification of phosphoproteins is reviewed. A new proteomic approach is described for the identification of the downstream targets of specific kinases that combines phosphorylation of cell lysates in in vitro kinase reactions by active recombinant kinase with protein separation by two-dimensional (2D) gel electrophoresis or SDS-PAGE and phosphoprotein identification by MALDI-TOF mass spectrometry or by phosphopeptide enrichment and tandem mass spectrometry. The results suggested that a combination of multiple approaches will be required to fully identify phosphoproteomes.     

Protein kinase C and mitogen-activated protein kinase signalling in oligodendrocytes

Oligodendrocytes (OL) play a significant physiological role in the central nervous system by creating the myelin sheath that allows for the efficient conduction of nerve impulses. Therefore, it is important to understand which signalling cascades define the proliferation, differentiation, survival, and myelin formation potential of these cells. Currently, much of the knowledge in this field has focused on two sets of protein kinase signalling molecules: Protein kinase C (PKC) and the mitogen-activated protein kinases (MAPKs). The roles of these kinases in OL are complex, and appear to be highly dependent on the developmental stage of the OL. Even so, some broad conclusions can be drawn from the multitude of experiments conducted on the roles of PKC and MAPKs in OL. For instance, PKC appears to have a proliferative effect on immature OL, while at the same time having an inhibitory effect on OL differentiation. In mature OL, the effects of PKC include increased process extension and myelin formation. The extracellular signal-regulated (ERK) members of the MAPK family also appear to increase process extensions in mature OL. On the other hand, the c-Jun N-terminal kinase (JNK) and p38 kinase members of the MAPK family appear to regulate apoptotic events in OL.     

Computerized histomorphometric study of the splenic collagen polymorphism: A control‐tissue for polarization microscopy

Previous articles have pointed out the presence of type III collagen within the extracellular structure of the parenchymatous organs. This study aimed to quantitatively characterize the collagen polymorphism at the capsule and parenchymal trabeculae of the largest lymphoid organ of the body i.e., the spleen, in mouse, rat, and rabbit models. Following a Picrosirius Red‐Polarization procedure and computer assisted image analysis of paraffin sections, the results showed (1) a predominant and significantly higher amount of type III collagen in the trabeculae area compared to the capsule area in the three species, (2) no statistical difference among the three species concerning the parenchymal collagen polymorphism or the type I/type III collagen ratio, (3) a heterogeneous type I/type III collagen ratio varying from 0.86 (mouse) to 6.62 (rabbit) in the fibromuscular capsule region. A qualitative analysis corroborated these histomorphometric results. In conclusion, the spleen may be used as (1) a control tissue to qualitatively visualize type I and III collagen under polarization microscopy and to validate the quality of PSR staining (2) an aid to accurately calibrate the angle of polarization before quantitative measurements of type I and type III collagen. Among the studied species, the rabbit spleen appeared to be the most appropriate control tissue as it showed the highest amount of type I collagen in the capsule and a similarly high amount of type III collagen in the parenchymal trabeculae. Microsc. Res. Tech. 78:900–907, 2015. © 2015 Wiley Periodicals, Inc.     

Peripheral synaptic contacts at mechanoreceptors in arachnids and crustaceans: morphological and immunocytochemical characteristics

Two types of sensory organs in crustaceans and arachnids, the various mechanoreceptors of spiders and the crustacean muscle receptor organs (MRO), receive extensive efferent synaptic innervation in the periphery. Although the two sensory systems are quite different-the MRO is a muscle stretch receptor while most spider mechanoreceptors are cuticular sensilla-this innervation exhibits marked similarities. Detailed ultrastructural investigations of the synaptic contacts along the mechanosensitive neurons of a spider slit sense organ reveal four important features, all having remarkable resemblances to the synaptic innervation at the MRO: (1) The mechanosensory neurons are accompanied by several fine fibers of central origin, which are presynaptic upon the mechanoreceptors. Efferent control of sensory function has only recently been confirmed electrophysiologically for the peripheral innervation of spider slit sensilla. (2) Different microcircuit configuration types, identified on the basis of the structural organization of their synapses. (3) Synaptic contacts, not only upon the sensory neurons but also between the efferent fibers themselves. (4) Two identified neurotransmitter candidates, GABA and glutamate. Physiological evidence for GABAergic and glutamatergic transmission is incomplete at spider sensilla. Given that the sensory neurons are quite different in their location and origin, these parallels are most likely convergent. Although their significance is only partially understood, mostly from work on the MRO, the close similarities seem to reflect functional constraints on the organization of efferent pathways in the brain and in the periphery.     

Mechanisms of fluid-flow-induced matrix production in bone tissue engineering

Matrix production by tissue-engineered bone is enhanced when the growing tissue is subjected to mechanical forces and/or fluid flow in bioreactor culture. Cells deposit collagen and mineral, depending upon the mechanical loading that they receive. However, the molecular mechanisms of flow-induced signal transduction in bone are poorly understood. The hyaluronan (HA) glycocalyx has been proposed as a potential mediator of mechanical forces in bone. Using a parallel-plate flow chamber the effects of removal of HA on flow-induced collagen production and NF-kappaB activation in MLO-A5 osteoid osteocytes were investigated. Short periods of fluid flow significantly increased collagen production and induced translocation of the NF-kappaB subunit p65 to the cell's nuclei in 65 per cent of the cell population. Enzymatic removal of the HA coat and antibody blocking of CD44 (a transmembrane protein that binds to HA) eliminated the fluid-flow-induced increase in collagen production but had no effect on the translocation of p65. HA and CD44 appear to play roles in transducing the flow signals that modulate collagen production over long-term culture but not in the short-term flow-induced activation of NF-kappaB, implying that multiple signalling events are initiated from the commencement of flow. Understanding the mechanotransduction events that enable fluid flow to stimulate bone matrix production will allow the optimization of bioreactor design and flow profiles for bone tissue engineering.     

Quantitative mass spectrometry to investigate epidermal growth factor receptor phosphorylation dynamics

Identifying proteins of signaling networks has received much attention, because an array of biological processes are entirely dependent on protein cross-talk and protein-protein interactions. Protein posttranslational modifications (PTM) add an additional layer of complexity, resulting in complex signaling networks. Of particular interest to our working group are the signaling networks of epidermal growth factor (EGF) receptor, a transmembrane receptor tyrosine kinase involved in various cellular processes, including cell proliferation, differentiation, and survival. Ligand binding to the N-terminal residue of the extracellular domain of EGF receptor induces conformational changes, dimerization, and (auto)-phosphorylation of intracellular tyrosine residues. In addition, activated EGF receptor may positively affect survival pathways, and thus determines the pathways for tumor growth and progression. Notably, in many human malignancies exaggerated EGF receptor activities are commonly observed. An understanding of the mechanism that results in aberrant phosphorylation of EGF receptor tyrosine residues and derived signaling cascades is crucial for an understanding of molecular mechanisms in cancer development. Here, we summarize recent labeling methods and discuss the difficulties in quantitative MS-based phosphorylation assays to probe for receptor tyrosine kinase (RTK) activity. We also review recent advances in sample preparation to investigate membrane-bound RTKs, MS-based detection of phosphopeptides, and the diligent use of different quantitative methods for protein labeling.     

两种油菜素内酯的多级串联质谱分析

The fragmentation pathways of 28-homobrassinolide(28-h-BL) and 28-epihomobrassinolide(28-eh-BL) were investigated by tandem mass spectrometry(MSⁿ, n＝1, 2, 3, 4). The proposed fragmentation mechanism could be potentially used to identify and distinguish brassinosteroids and their isomers.     

rb-bFGF联合激光治疗痤疮凹陷性瘢痕的效果观察

目的:探讨重组牛碱性成纤维细胞生长因子凝胶(rb-bFGF)联合点阵超脉冲二氧化碳(CO₃)激光治疗痤疮凹陷性瘢痕患者的效果。方法:将入组的86例痤疮凹陷性瘢痕患者随机分为2组,即对照组和观察组各43例,对照组给予点阵超脉冲CO₃激光治疗,观察组基于以上加予rb-bFGF治疗,对比两组痤疮瘢痕权重评分(ECCA)、主诉症状视觉模拟评分(VAS)、水肿持续时间、脱痂时间及炎症性色素沉着(PIH)持续时间、治疗前后的皮肤生理指标[经皮水份丢失(TEWL)、pH值、角质层含水量、皮肤红斑情况(a值)]、治疗前后的血清p38MAPK通路蛋白相关指标[重组人丝裂原活化激酶1(MEK1)、重组人丝裂原活化激酶2(MEK2)、细胞外信号调节激酶1(ERK1)及细胞外信号调节激酶2(ERK2)]和治疗期间的不良反应。结果对比两组治疗后ECCA评分、VAS评分,观察组显著低于对照组(P<0.05);对比两组水肿持续时间、脱痂时间及PIH持续时间,观察组显著短于对照组(P<0.05);对比两组治疗前后TEWL值、pH值及a值,观察组显著低于治疗前及对照组(P<0.05),对比两组治疗前后角质层含水量,观察组显著高于治疗前及对照组(P<0.05),以上指标对照组治疗前后差异无统计学意义(P>0.05)。对比两组治疗前后血清MEK1、MEK2、ERK1及MEK2水平,两组显著低于治疗前(P<0.05),且观察组显著低于对照组(P<0.05);观察组的不良反应总发生率显著低于对照组(9.30%vs 25.58%,P<0.05)。结论:rb-bFGF辅助点阵超脉冲CO₃激光治疗痤疮凹陷性瘢痕,能明显促进术后创伤愈合,改善皮肤生理状态,减轻皮肤炎性损伤,减少不良反应。