共查询到20条相似文献,搜索用时 31 毫秒
1.
Chen H Gu DN Burton-Wurster N MacLeod JN 《The Journal of biological chemistry》2002,277(22):20095-20103
The cartilage-specific (V + C)(-) fibronectin isoform does not efficiently heterodimerize with other V-region splice variants of fibronectin. To understand better the structural elements that determine this restricted dimerization profile, a series of truncated fibronectin expression constructs with various internal deletions in the V, III-15, or I-10 segments were constructed and co-transfected into COS-7 cells with either the V(+)C(+) or the (V + C)(-) isoform. SDS-PAGE and immunoblot analyses of the resulting conditioned media suggest that the I-10 segment must either be present in both monomeric subunits of fibronectin or absent from both subunits for efficient dimerization to occur. Further studies suggest that the I-10 segment specifically, not simply a balanced number of type I repeats at the carboxyl terminus of each monomeric subunit, plays an important role in determining different fibronectin dimerization patterns. Neither I-11 nor I-12 could be substituted for segment I-10 without significantly reducing the formation of heterodimers. Therefore, absence of segment I-10 explains why (V + C)(-) fibronectin is not found in heterodimeric configurations with other native V-region splice variants in cartilage. The unique dimerization pattern of (V + C)(-) fibronectin does not prevent matrix formation yet is consistent with this isoform having specialized properties in situ that are important for either the structural organization and biomechanical properties of cartilage or the regulation of a chondrocytic phenotype. 相似文献
2.
Nancy Burton-Wurster Hao Chen Rina Gendelman Michael L Jackson Lucille F Gagliardo Da Nian Gu Russell R Zelko George Lust James N MacLeod 《Matrix biology》2002,21(5):393-398
The population of fibronectins in adult mammalian cartilage includes high levels of a cartilage-specific (V+C)(-) isoform which lacks the V, III-15, and I-10 segments and thus contains a novel junction between protein segments III-14 and I-11. We report production of a monoclonal antibody specific for (V+C)(-) fibronectin without cross-recognition of V(+)C(+) and V(-)C(+) isoforms found in plasma and other tissues. Presentation of epitope to this antibody requires the III-14/I-11 junction, but the epitope itself extends beyond 14 amino acids immediately surrounding the junction site and involves a conformational change in III-14 and/or the N-terminal portion of I-11. The antibody, designated Mab 5D10 anti (V+C)(-), displays specificity for (V+C)(-) fibronectin from multiple mammalian species including humans and utility in immunoblots, immunohistochemistry, and ELISA. 相似文献
3.
Decorin modulates collagen I-stimulated, but not fibronectin-stimulated, migration of C2C12 myoblasts 总被引:1,自引:0,他引:1
Extracellular matrix factors, specifically fibronectin and collagen I, are essential for structural support during muscle regeneration. Decorin has been identified as an anti-fibrotic agent with binding sites located on both fibronectin and collagen I. Upon injury, activated myoblasts are required to migrate through the extracellular matrix factors deposited by the myofibroblasts to facilitate skeletal muscle regeneration. In this study we looked at the effects decorin on fibronectin- and collagen I-stimulated myoblast migration. Dose response studies demonstrated 10 μg/ml, 5 μg/ml and 25 μg/ml as the optimal stimulatory concentrations of decorin (1.2 fold increase), fibronectin (3.5 fold increase) and collagen I (2.4 fold increase), when compared with control respectively. A synergistic effect was identified when decorin and collagen I were added in combination; this effect was not evident when decorin was added with fibronectin. The effects of these factors on the ROCK signalling pathway were also analyzed. ROCK-2 was identified as the key Rho-activated kinase isoform involved in migration, due to its higher expression levels and localisation to focal points within migrating C2C12 myoblasts. Decorin and collagen I in combination stimulated an increase in the number of ROCK-2 localized focal points when compared with control, decorin and collagen I added separately. Fibronectin did not show any increase in ROCK-2 focal points when compared with control. These results show for the first time that decorin can modify collagen I-stimulated, but not fibronectin-stimulated myoblast migration in vitro. Furthermore, the synergistic, rather than additive, effect observed suggests a direct modification of collagen I signalling by decorin mediated, at least in part, by ROCK-2 rather than ROCK-1. 相似文献
4.
Lipoamide dehydrogenase catalyses the NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-(2)H]-NADH was studied anaerobically at 4 degrees C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (k(for) = 1260 s(-)(1), k(rev) = 590 s(-)(1)) and represents the formation of FADH(2).NAD(+), an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [(D)(k(for) + k(rev)) approximately 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (k(obs) = 200 s(-)(1)). This phase represents the intramolecular transfer of electrons from FADH(2) to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH(2). The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH(4). The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (k(for) = 57 s(-)(1), k(rev) = 128 s(-)(1)) isomerization of EH(2) or slow release of NAD(+) before rapid NADH binding and reaction to form EH(4). The mechanism of oxidation of EH(2) by NAD(+) was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH(2) shifted to 600 nm upon NAD(+) binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH(2).NAD(+) complex. This was followed by two phases. The first phase (k(obs) = 750 s(-)(1)), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH(2).NAD(+). The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of approximately 115 s(-)(1) probably represents the overall rate of formation of E(ox) and NADH from EH(2) and NAD(+), and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation. 相似文献
5.
Khanna-Chopra R Sabarinath S 《Biochemical and biophysical research communications》2004,320(4):1187-1192
Chenopodium murale is a weed species having wide adaptation to different climatic regimes and experiences a temperature range of 5-45 degrees C during its life span. Higher temperatures may result in heat stress, which induces higher ROS production leading to oxidative stress in the plant. Superoxide dismutase enzyme (SOD, EC.1.15.1.1) is ubiquitous, being widely distributed among O(2)(-) consuming organisms and is the first line of defense against oxidative stress. In this study, we have characterized the thermostability of the SOD isozymes from C. murale in vitro. The leaf protein extracts, thylakoidal and stromal fractions were subjected to elevated temperatures ranging from 50 degrees C to boiling and analyzed for activity and isoform pattern of SOD. Out of six SOD isoforms, SOD V showed stability even after boiling the extract for 10min. Under high temperature treatment (>60 degrees C) there was an appearance of a new SOD band with higher electrophoretic mobility. The inhibitor studies and subcellular analysis revealed that the SOD V isoform was a chloroplastic Cu/Zn SOD. The stromal Cu/Zn SOD (SOD V) was more stable than the co-migrating thylakoidal isozyme at 80 degrees C and boiling for 10min. Hence, we report an unusual, constitutive thermostable chloroplastic Cu/Zn SOD from C. murale, which may contribute towards its heat tolerance. 相似文献
6.
Simard CF Daigle ND Bergeron MJ Brunet GM Caron L Noël M Montminy V Isenring P 《The Journal of biological chemistry》2004,279(46):48449-48456
The first isoform of the Na(+)-K(+)-Cl(-) cotransporter (NKCC1) is of central importance for the control of cellular ion concentration and epithelium-mediated salt secretion. Several studies have established that a change in intracellular [Cl(-)] (Cl(-)(i)) represents a key signaling mechanism by which NKCC1-induced Cl(-) movement is autoregulated and by which Cl(-) entry and exit on opposite sides of polarized cells are coordinated. Although this signaling mechanism is coupled to a pathway that leads to post-translational modification of the carrier, no unifying model currently accounts for the ion dependence of NKCC1 regulation. In this paper, evidence is presented for the first time that hsp90 associates with the cytosolic C terminus of NKCC1, probably when the carrier is predominantly in its unfolded form during early biogenesis. Evidence is also presented that the Cl(-)(i)-dependent regulatory pathway can be activated by a thermal stress but that it is no longer operational if NKCC1-expressing cells are pretreated with geldanamycin, an antibiotic that inhibits hsp90, albeit nonspecifically. Taken together, our data indicate that binding of hsp90 to NKCC1 may be required for Na(+)-K(+)-Cl(-) cotransport to occur at the cell surface and that it could play an important role in ion-dependent signaling mechanisms, insofar as the maneuvers that were used to alter the expression or activity of the chaperone do not exert their main effect by inducing other cellular events such as the unfolded protein response. Further studies will be required to elucidate the functional relevance of this novel interaction. 相似文献
7.
Kinsella MG Fischer JW Mason DP Wight TN 《The Journal of biological chemistry》2000,275(18):13924-13932
Decorin is a member of the widely expressed family of small leucine-rich proteoglycans. In addition to a primary role as a modulator of extracellular matrix protein fibrillogenesis, decorin can inhibit the cellular response to growth factors. Decorin expression is induced in endothelial cells during angiogenesis, but not when migration and proliferation are stimulated. Thus, decorin may support the formation of the fibrillar pericellular matrix that stabilizes the differentiated endothelial phenotype during the later stages of angiogenesis. Therefore, we tested whether constitutive decorin expression alone could modify endothelial cell migration and proliferation or affect pericellular matrix formation. To this end, replication-defective retroviral vectors were used to stably express bovine decorin, which was detected by Northern and Western blotting. The migration of endothelial cells that express decorin is significantly inhibited in both monolayer outgrowth and microchemotaxis chamber assays. The inhibition of cell migration by decorin was not accompanied by decreased proliferation. In addition, endothelial cells that express decorin assemble an extensive fibrillar fibronectin matrix more rapidly than control cells as assessed by immunocytochemical and fibronectin fibrillogenesis assays. These observations suggest that cell migration may be modulated by the influence of decorin on the assembly of the cell-associated extracellular matrix. 相似文献
8.
Evidence for a functional role for extracellular matrix (ECM) proteins in adipose tissue is demonstrated in dynamic changes in expression of ECM genes during adipocyte differentiation and in obesity. Components of the ECM may regulate adipose cell number expansion by restricting pre-adipocyte proliferation, regulating apoptosis and inhibiting adipogenesis. Although pre-adipocytes express multiple proteoglycans, their role in pre-adipocyte proliferation up to now has remained unknown. The study described here was conducted to characterize roles of small leucine-rich proteoglycans (SLRPs) in adipocyte proliferation. Pre-adipocytes were seeded on plates coated with biglycan and decorin and were allowed to differentiate. In addition, pre-adipocytes were incubated on plates coated with biglycan, decorin, or fibronectin and measurements were made of cell proliferation and apoptosis. We are able to report that SLRPs decorin and biglycan did not affect differentiation of our 3T3-L1 cells; however, biglycan and decorin did reduce proliferation of pre-adipocytes, partly by induction of apoptosis. Furthermore, anti-proliferative capabilities of decorin and biglycan were nullified with removal of GAG side-chains suggesting that the chains played key roles in anti-proliferative effects of the SLRPs. We also found that co-treatment of decorin or biglycan with the proteoglycan fibronectin restored normal proliferation, an indication that multiple ECM proteins may act in concert to regulate overall proliferation rates of pre-adipocytes. These studies indicate that SLRPs may compose a regulatory factor in adipose tissue expansion, through hyperplasia. 相似文献
9.
Formation and coherent propagation of nuclear wavepackets on potential energy surfaces of the excited state of the primary electron donor P and of the charge transfer states P(+)B(A)(-) and P(+)H(A)(-) were studied in native and pheophytin-modified Rhodobacter sphaeroides R-26 reaction centers (RCs) induced by 25 fs excitation (where B(A) and H(A) are the primary and secondary electron acceptors, respectively). The processes were monitored by measuring coherent oscillations in kinetics of the time evolution of the stimulated emission band of P at 935 nm, of the absorption band of B(A)(-) at 1020 nm, and of the bleaching band of H(A) at 760 nm. It was found that the nuclear wavepacket motion on the 130-140 cm(-1) surface of P is directly induced by light absorption in P. When the wavepacket approaches the intersection between P and P(+)B(A)(-) surfaces at 120 and 380 fs delays, the formation of intermediate mixed-state emitting light at 935 nm (P) and absorbing light at 1020 nm (P(+)B(A)(-)) takes place. At the latter time, the wavepacket is transferred to the 32 cm(-1) mode which can belong to the P hypersurface effectively transferring the wavepacket to the P(+)B(A)(-) surface or can represent a diabatic surface which is formed by the states P and P(+)B(A)(-). The wavepacket motion on the P(+)B(A)(-) surface or on the P(+)B(A)(-) part of the mixing surface is accompanied by irreversible electron transfer to H(A). This process is monitored by the kinetics of 1020 nm band development and 760 nm band bleaching (delayed with respect to 1020 nm band development) which both have the enhanced 32 cm(-1) mode in Fourier transform (FT) spectra. The mechanism of wavepacket transfer from the 130-140 cm(-1) to the 32 cm(-1) mode is discussed. 相似文献
10.
Sulochana KN Fan H Jois S Subramanian V Sun F Kini RM Ge R 《The Journal of biological chemistry》2005,280(30):27935-27948
Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 3(10) helix and beta-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs. 相似文献
11.
Growth on a decorin matrix results in decreased human airway smooth muscle cell (HASMC) number, by decreasing proliferation and increasing apoptosis. We questioned whether these effects were related to abnormal extracellular matrix (ECM)-cell adhesion. HASMCs were seeded on decorin, biglycan, collagen type I or plastic. Actin organization and focal adhesion formation were assessed by staining for filamentous (F) and globular (G) actin, and vinculin, respectively. Gene expression for focal adhesion proteins, ECM molecules and HASMC receptors was measured. Protein levels for fibronectin, α(2), α(5), α(v) and β(3) integrin subunits and, focal adhesion kinase (FAK) were assessed. F-actin filaments were prominent in cells seeded on collagen I and plastic, less apparent in cells cultured on biglycan and faint in cells on decorin. Vinculin clustering was decreased in cells seeded on decorin and biglycan, as was vinculin gene expression. Compared to cells on plastic, cells on decorin had an increase in fibronectin gene expression. Seeding on decorin caused an increase in α(2) integrin subunit and platelet-derived growth factor receptor A gene expression. There was also an increase in α(2) and α(v) integrin subunit protein. Finally, FAK protein levels in cells seeded on decorin or biglycan were decreased compared to cells seeded on plastic or collagen I. Cells grown on proteoglycan matrices demonstrate evidence of abnormalities during many of the key processes involved in normal cell adhesion. Upregulation of cell surface receptor proteins, such as α(2) integrin subunit, may represent a compensatory mechanism to overcome poor adhesion induced by growth on these matrices. 相似文献
12.
Lee JE Nam JH Kim SJ 《American journal of physiology. Gastrointestinal and liver physiology》2005,288(4):G822-G831
To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage (V(m)) and intracellular concentrations of Ca(2+), Na(+) ([Na(+)](c)), Cl(-), and H(+) (pH(i)) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 muM, 20 s), RSMGA showed strong delayed hyperpolarization (V(h,ACh); -95 +/- 1.8 mV) that was abolished by ouabain. In the HCO(3)(-)-free condition, the V(h,ACh) was also blocked by bumetanide, a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC). In the presence of HCO(3)(-) (24 meq, bubbled with 5% CO(2)), however, the V(h,ACh) was not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride (EIPA), a Na(+)/H(+) exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na(+)](c) was totally blocked by bumetanide in the absence of HCO(3)(-), but only by one-half in the presence of HCO(3)(-). ACh induced a prominent acidification of pH(i) in the presence of HCO(3)(-), and the acidification was further increased by EIPA treatment. Without HCO(3)(-), an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pH(i) during the application of NH(4)(+) (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO(3)(-). In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [Na(+)](c). The intracellular Na(+)-induced activation of electrogenic Na(+)/K(+)-ATPase causes V(h,ACh). The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO(3)(-). 相似文献
13.
Emoto M Miyamoto M Yoshizawa I Emoto Y Schaible UE Kita E Kaufmann SH 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(3):1426-1432
Although macrophages play a central role in the pathogenesis of septic shock, NK1(+) cells have also been implicated. NK1(+) cells comprise two major populations, namely NK cells and V alpha 14(+)NKT cells. To assess the relative contributions of these NK1(+) cells to LPS-induced shock, we compared the susceptibility to LPS-induced shock of beta(2)-microglobulin (beta(2)m)(-/-) mice that are devoid of V alpha 14(+)NKT cells, but not NK cells, with that of wild-type (WT) mice. The results show that beta(2)m(-/-) mice were more susceptible to LPS-induced shock than WT mice. Serum levels of IFN-gamma following LPS challenge were significantly higher in beta(2)m(-/-) mice, and endogenous IFN-gamma neutralization or in vivo depletion of NK1(+) cells rescued beta(2)m(-/-) mice from lethal effects of LPS. Intracellular cytokine staining revealed that NK cells were major IFN-gamma producers. The J alpha 281(-/-) mice that are exclusively devoid of V alpha 14(+)NKT cells were slightly more susceptible to LPS-induced shock than heterozygous littermates. Hence, LPS-induced shock can be induced in the absence of V alpha 14(+)NKT cells and IFN-gamma from NK cells is involved in this mechanism. In WT mice, hierarchic contribution of different cell populations appears likely. 相似文献
14.
The NH(4)(+) and NO(3)(-) uptake kinetics by Typha latifolia L. were studied after prolonged hydroponics growth at constant pH 3.5, 5.0, 6.5 or 7.0 and with NH(4)(+) or NO(3)(-) as the sole N-source. In addition, the effects of pH and N source on H(+) extrusion and adenine nucleotide content were examined. Typha latifolia was able to grow with both N sources at near neutral pH levels, but the plants had higher relative growth rates, higher tissue concentrations of the major nutrients, higher contents of adenine nucleotides, and higher affinity for uptake of inorganic nitrogen when grown on NH(4)(+). Growth almost completely stopped at pH 3.5, irrespective of N source, probably as a consequence of pH effects on plasma membrane integrity and H(+) influx into the root cells. Tissue concentrations of the major nutrients and adenine nucleotides were severely reduced at low pH, and the uptake capacity for inorganic nitrogen was low, and more so for NO(3)(-)-fed than for NH(4)(+)-fed plants. The maximum uptake rate, V(max), was highest for NH(4)(+) at pH 6.5 (30.9 micro mol h(-1) g(-1) root dry weight) and for NO(3)(-) at pH 5.0 (31.7 micro mol h(-1) g(-1) root dry weight), and less than 10% of these values at pH 3.5. The affinity for uptake as estimated by the half saturation constant, K((1/2)), was lowest at low pH for NH(4)(+) and at high pH for NO(3)(-). The changes in V(max) and K((1/2)) were thus consistent with the theory of increasing competition between cations and H(+) at low pH and between anions and OH(-) at high pH. C(min) was independent of pH, but slightly higher for NO(3)(-) than for NH(4)(+) (C(min)(NH(4)(+)) approximately 0.8 mmol m(-3); C(min)(NO(3)(-)) approximately 2.8 mmol m(-3)). The growth inhibition at low pH was probably due to a reduced nutrient uptake and a consequential limitation of growth by nutrient stress. Typha latifolia seems to be well adapted to growth in wetland soils where NH(4)(+) is the prevailing nitrogen compound, but very low pH levels around the roots are very stressful for the plant. The common occurrence of T. latifolia in very acidic areas is probably only possible because of the plant's ability to modify pH-conditions in the rhizosphere. 相似文献
15.
Valeria Rivarola Paula Ford María del Pilar Flamenco Luciano Galizia Claudia Capurro 《Cellular physiology and biochemistry》2007,20(5):549-558
Arginine-vasopressin (AVP) has been proposed to be involved in the modulation of acid-base transporters; however, the nature of the mechanisms underlying AVP direct action on intracellular pH (pH(i)) in the cortical collecting duct (CCD) is not yet clearly defined. The aim of the present study was to elucidate which are the proteins implicated in AVP modulation of pH(i), as well as the receptors involved in these responses using a CCD cell line (RCCD(1)); pH(i) was monitored with the fluorescent dye BCECF in basal conditions and after stimulation with basolateral 10(-8) M AVP. Specific V1- or V2-receptor antagonists were also used. RT-PCR studies demonstrated that RCCD(1) cells express V1a and V2 receptors. Functional studies showed that while V2-receptor activation induced a biphasic response (alkalinization-acidification), V1-receptor activation resulted in an intracellular acidification. The V2-mediated alkalinization phase involves the activation of basolateral NHE-1 isoform of the Na(+)/H(+) exchanger while in the acidification phase CFTR is probably implicated. On the other hand, V1-mediated acidification was due to activation of a Cl(-)/HCO(3)(-) exchanger. We conclude that in RCCD(1) cells AVP selectively activates, via a complex of V1 and V2 receptor-mediated actions, different ion transporters linked to pH(i) regulation which might have physiological implications. 相似文献
16.
Mice lacking serum amyloid P component do not necessarily develop severe autoimmune disease 总被引:4,自引:0,他引:4
Soma M Tamaoki T Kawano H Ito S Sakamoto M Okada Y Ozaki Y Kanba S Hamada Y Ishihara T Maeda S 《Biochemical and biophysical research communications》2001,281(1):200-205
Interleukin-10 (IL-10) is a cytokine with many regulatory functions. In particular, IL-10 exerts neutralizing effect on other cytokines, and therefore IL-10 is thought to have important therapeutic implications. Recent reports suggest that IL-10 regulates not only immunocytes but also collagen and collagenase gene expression in fibroblasts. In this study, we investigated the effect of IL-10 on gene expression of extracellular matrix (ECM) proteins, such as type I collagen, fibronectin, and decorin, in human skin fibroblasts. Results of Northern blot analysis showed that both collagen I and fibronectin mRNAs were downregulated, while decorin gene expression was enhanced by IL-10 (10 ng/ml) time-dependently (6-24 h). alpha1(I) collagen and fibronectin mRNAs were decreased to one-third and one-fourth, respectively, by 50 ng/ml IL-10, whereas decorin mRNA was increased up to 2.7-fold by 50 ng/ml IL-10. Response to IL-10 by scleroderma fibroblasts was similar to that in normal dermal fibroblasts, with decreased expression levels of collagen and fibronectin and induced decorin mRNA levels. Transforming growth factor-beta (TGF-beta) is a crucial fibrogenic cytokine which upregulates the mRNA expression of collagen and fibronectin, whereas it downregulates decorin mRNA expression in fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) has recently been shown to upregulate the type I collagen mRNA expression in cultured fibroblasts. We therefore examined whether IL-10 alters gene expression of ECM elicited by TGF-beta and MCP-1. Our results demonstrated that IL-10 downregulated the TGF-beta-elicited increase of mRNA expression of type I collagen and fibronectin, while partially recovering TGF-beta-elicited decrease of decorin expression in normal skin fibroblasts. By contrast, IL-10 did not alter the MCP-1-elicited upregulation of mRNA expression of either alpha1(I) collagen and decorin. Our data indicate that IL-10 differentially regulates TGF-beta and MCP-1 in the modulation of ECM proteins and therefore suggest that IL-10 plays a role in the regulation of tissue remodeling. 相似文献
17.
Influence of decorin on fibroblast adhesion to fibronectin. 总被引:8,自引:0,他引:8
Decorin is a ubiquitous small dermatan sulfate proteoglycan carrying a single glycosaminoglycan chain. It is known for its ability to bind, via its core protein, to interstitial collagens. Decorin was purified from the secretions of cultured human skin fibroblasts under non-denaturing conditions. The intact proteoglycan and its glycosaminoglycan-free core protein were tested for their interference with fibroblast adhesion to a fibronectin substrate. Concentrations of 40 nmoles or more of hexuronic acid/ml of decorin or equivalent amounts of core protein inhibited cell adhesion. Inhibition was caused by an interaction of core protein with fibronectin and not by masking of the fibronectin receptor. When cell-binding fragments of fibronectin were used as substrates, a similar inhibition of cell adhesion by decorin core protein was found, and in vitro assays demonstrated an interaction of core protein with the cell-binding domain of fibronectin. Decorin core protein also inhibited the low degree of cell adhesion to heparin-binding fragments on the N-terminus and near the C-terminus of the fibronectin molecules. 相似文献
18.
Vial C Gutiérrez J Santander C Cabrera D Brandan E 《The Journal of biological chemistry》2011,286(27):24242-24252
Fibrotic disorders are the end point of many chronic diseases in different tissues, where an accumulation of the extracellular matrix occurs, mainly because of the action of the connective tissue growth factor (CTGF/CCN2). Little is known about how this growth factor activity is regulated. We found that decorin null myoblasts are more sensitive to CTGF than wild type myoblasts, as evaluated by the accumulation of fibronectin or collagen III. Decorin added exogenously negatively regulated CTGF pro-fibrotic activity and the induction of actin stress fibers. Using co-immunoprecipitation and in vitro interaction assays, decorin and CTGF were shown to interact in a saturable manner with a K(d) of 4.4 nM. This interaction requires the core protein of decorin. Experiments using the deletion mutant decorin indicated that the leucine-rich repeats (LRR) 10-12 are important for the interaction with CTGF and the negative regulation of the cytokine activity, moreover, a peptide derived from the LRR12 was able to inhibit CTGF-decorin complex formation and CTGF activity. Finally, we showed that CTGF specifically induced the synthesis of decorin, suggesting a mechanism of autoregulation. These results suggest that decorin interacts with CTGF and regulates its biological activity. 相似文献
19.
Intact fibronectin (FN) protects cells from apoptosis. When FN is fragmented, specific domains induce proteinase expression in fibroblasts. However, it is not known whether specific domains of FN can also regulate apoptosis. We exposed fibroblasts to four recombinant FN fragments and then assayed for apoptosis using criteria of cellular shape change, condensed nuclear morphology, and DNA fragmentation. The fragments extended from the RGD-containing repeat III10 to III15; they included (V(+)) or excluded (V(-)) the alternatively spliced V region and contained either a mutated (H(-)) or an unmutated (H(+)) heparin binding domain. Only the V(+)H(-) fragment triggered decreases in pp125(FAK) levels and apoptosis, which was rescued by intact FN and inhibitors of caspase-1 and caspase-3. This apoptotic mechanism was mediated by a chondroitin sulfate proteoglycan, since treating cells with chondroitin sulfate or chondroitinase reversed the apoptotic cell shape changes. The alpha4 integrin receptor may also be involved, since using a blocking antibody to alpha4 alone induced apoptotic cell shape changes, whereas co-treatment with this antibody plus V(+)H(+) reversed these effects. These results demonstrate that the V and heparin binding domains of FN modulate pp125(FAK) levels and regulate apoptosis through a chondroitin sulfate proteoglycan- and possibly alpha4 integrin-mediated pathway, which triggers a caspase cascade. 相似文献
20.
Diaz-Sylvester PL Fiori MC Dieguez SM Müller AC Lopardo ML Amorena CE 《American journal of physiology. Regulatory, integrative and comparative physiology》2008,294(6):R2014-R2020
The acute effect of angiotensin-converting enzyme inhibition (ACEi) on proximal convoluted tubule (PCT) function is well documented. However, the effect of chronic treatment is less known. The aim of this work was to evaluate the effect of chronic ACEi on PCT acidification (J(HCO(3)(-))). Rats received enalapril (10 mg.kg(-1).day(-1), added to the drinking water) during 3 mo. Micropuncture experiments were performed to measure the effect of chronic ACEi on J(HCO(3)(-)). Nitric oxide (NO.) synthesis in kidney cortex homogenates was assessed by quantifying the conversion of [(14)C]-L-arginine to [(14)C]-L-citrulline. Western blot analysis was performed to determine the abundances of V-H(+)ATPase and NHE3 isoform of the Na(+)/H(+) exchanger in proximal brush-border membrane vesicles (BBMV). Enalapril treatment induced an approximately 50% increase in J(HCO(3)(-)). Luminal perfusion with ethyl-isopropyl amiloride (EIPA) 10(-4)M or bafilomycin 10(-6)M decreased J(HCO(3)(-)) by approximately 60% and approximately 30%, respectively, in both control and enalapril-treated rats. The effect of EIPA and bafilomycin on absolute J(HCO(3)(-)) was larger in enalapril-treated than in control rats. Acute inhibition of NO. synthesis with N(G)-nitro-L-arginine methyl ester abolished the enalapril-induced increase in J(HCO(3)(-)). Cortex homogenates from enalapril-treated rats displayed a 46% increase in nitric oxide synthase (NOS) activity compared with those from untreated animals. Enalapril treatment did not affect the abundances of NHE3 and V-H(+)ATPase in BBMV. Our results suggest that PCT acidification is increased during chronic ACEi probably due to an increase in NO. synthesis, which would stimulate Na(+)/H(+) exchange and electrogenic proton transport. 相似文献