Decreased PDGF Receptor Kinase Activity in Fibroblasts Contracting Stressed Collagen Matrices

Fibroblasts cultured in mechanically stressed collagen matrices proliferate, whereas cells in floating collagen matrices become quiescent. Previous research indicated that one factor contributing to cell quiescence in floating matrices was reduced receptor autophosphorylation in response to PDGF stimulation (i.e., PDGF receptor desensitization). To learn more about the mechanism of PDGF receptor desensitization, we analyzed changes in PDGF receptor autophosphorylation and receptor kinase activity after stressed collagen matrices were switched to floating conditions, which results in rapid cell contraction and dissipation of mechanical stress. PDGF receptor desensitization occurred during contraction stimulated by serum but not in the absence of serum, and desensitization was prevented by inhibitors of contraction but not by inhibitors of the contraction-activated cyclic AMP signaling pathway. Receptor desensitization resulted from decreased receptor kinase activity rather than from elevated protein tyrosine phosphatase activity, and only receptors unoccupied at the time of contraction were affected. After contraction, radiolabeled PDGF binding to the cells was decreased, which suggested that receptor desensitization resulted from a contraction-dependent change in receptor availability or affinity.     

Deregulation of Collagen Phagocytosis in Aging Human Fibroblasts: Effects of Integrin Expression and Cell Cycle

Intracellular degradation of collagen by phagocytosis in fibroblasts is essential for physiological remodeling of the extracellular matrix in a wide variety of connective tissues but imbalances between degradation and synthesis can lead to loss of tissue collagen. As aging is associated with loss of dermal and periodontal collagen and with increased lysomomal enzyme content in fibroblasts, we examined the regulation of collagen phagocytosis by integrin expression and the cell cycle in anin vitrofibroblast aging model. Two different fibroblast lines (CL1; CL2) at the fourth subculture were passaged up to replicative senescence to model aging processesin vitro.Cells were incubated with collagen-coated or BSA-coated green fluorescent beads for 3 h to assess α₂β₁-integrin-mediated or nonspecific phagocytosis, respectively. Single-cell suspensions were stained with DAPI and sulforhodamine 101 to separate cycling G₁and noncycling G₀cells. Staining for α₂-integrin, bead internalization, and bivariate analyses of DNA/protein content were measured by three-color flow cytometry. Serum deprivation was used to induce increases in the proportion of G₀cells. For G₁cells, the proportion of collagen phagocytic cells was >50% for all passages and collagen beads were internalized >5-fold more frequently than BSA beads. In contrast, G₀cells with diploid DNA content but low protein content exhibited greatly reduced phagocytic capacity (<10% of cells internalized collagen or BSA beads), the number of beads per cells was 4-fold less, and α₂integrin expression was very low compared to G₁cells. The proportion of collagen phagocytic cells and the proportion of α₂-integrin-positive cells increased with transit through the cell cycle. At higher passage numbers mean cell volume and cytoplasmic granularity were reduced 30% but at replicative senescence cells with large surface area and subdiploid DNA predominated. The proportion of collagen and BSA phagocytic G₁cells increased 1.5- and 5-fold, respectively, and the number of beads per cell increased <3-fold. However, surface α₂-integrin staining remained unchanged. These data indicate that the collagen and nonspecific internalization pathways were greatly upregulated, independent of cell cycle phase, and that cellular agingin vitrostrongly influences the specificity and rate of phagocytic processes in fibroblasts. We suggest that age-related loss of collagen in connective tissues undergoing turnover may be a manifestation of a deregulated increase of collagen phagocytosis in which the net loss of degraded collagen exceeds new synthesis.     

Increased levels of c-fos and c-myc mRNA in ATP-stimulated endothelial cells

In bovine aortic endothelial cells, ATP induced a transient and sequential accumulation of c-fos and c-myc mRNA, which was detected after 1 hour and 3 hours, respectively. The effect of ATP on c-fos mRNA was stronger than that of TNF and bFGF. Both ATP and bFGF increased c-myc mRNA after a 3 hour treatment, whereas TNF did not. If none of the 3 agonists tested induced a selective expression of c-fos or c-myc, each of them was associated with a different quantitative combination of the 2 signals, which might be related to the distinct responses that they trigger in endothelial cells.     

Expression of Retinoid X Receptors in Human Dermal Fibroblasts

Retinoic acid (RA) is known to exert profound effects on growth and differentiation in human dermal fibroblasts. In the observations presented here, we examined the regulation of expression of members of the RXR multigene family in human dermal fibroblasts. We showed that the messenger RNAs for both RXRα and RXRβ are expressed in human fibroblasts, but that the messenger RNA for RXRγ is not detectable in these cells. Electrophoretic mobility shift studies of binding to the β2RARE in human dermal fibroblasts demonstrated that a single complex binds to β2RARE in the absence of RA. Stimulating cells with all-transRA induced a second complex. An antibody to the RXRβ protein supershifted both complexes, while an antibody to the RXRα S/B protein had no effect on the binding. These data demonstrate that RXRβ plays an important role in retinoid-regulated signal transduction pathways in human dermal fibroblasts and the regulation of expression of the RXR gene family is different from that of the RAR gene family.     

Induction of c-fos mRNA Expression in Rat Striatum by Neuroleptic Drugs

The effect of selective dopamine D2 receptor-acting drugs on striatal c-fos mRNA expression in the rat has been investigated by Northern hybridization and autoradiography to determine a possible role for c-fos in the initiation of adaptive changes in D2 receptor number by neuroleptic drugs. The neuroleptic drug haloperidol, a D2 receptor antagonist, was found to produce a rapid and transient induction of c-fos mRNA expression as compared with the expression in animals treated with saline. This induction by haloperidol was found to be dose dependent and D2 receptor mediated, inasmuch as a D2 agonist completely reversed the induction and the inactive isomer of the neuroleptic butaclamol, which does not produce an increase in D2 receptors, had no effect on c-fos mRNA expression. From these data, it can be concluded that c-fos expression in striatum is under dopamine D2 receptor-mediated inhibitory control. It is suggested that c-fos may play a role in the initiation of the increase in D2 receptor number produced by chronic neuroleptic drug treatment.     

Increased Adipogenesis of Human Adipose-Derived Stem Cells on Polycaprolactone Fiber Matrices

With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells - differentiated to adipocytes in vitro - are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics – studied by coherent anti-Stokes Raman scattering microscopy - to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.     

Styrene Altered Clock Gene Expression in Serum-Shocked Cultured Human Fibroblasts

The circadian clock can regulate the metabolic process of xenobiotics, but little is known as to circadian rhythms can be perturbed by xenobiotics. Styrene is a organic chemical widely used in occupational settings. The effects of styrene on the circadian genes of HuDE cells were evaluated after serum-shocking synchronization. A subtoxic dose of 100 µM of styrene altered the expression of clock genes BMAL1, PER2, PER3, CRY1, CRY2, and REV-ERB-α.     

Factors in Bovine Colostrum that enhance the Migration of Human Fibroblasts in Type I Collagen Gels

Bovine colostrum has an activity that increases the migration of WI38 fibroblasts. We evaluated the motility of fibroblasts by their ability to contract collagen gels. Part of the activity was absorbed by anion-exchange chromatography at pH 6.4, and eluted by 0.2-0.3 M sodium chloride. The activity was separated into many fractions corresponding to 20-150 kDa by gel filtration chromatography under acidic conditions. The major peak of the activity coincided with 50-70 kDa.     

Cyclooxygenase-3 Gene Expression in Alzheimer Hippocampus and in Stressed Human Neural Cells

The cyclooxygenase (COX) superfamily of prostaglandin synthase genes encode a constitutively expressed COX-1, an inducible, highly regulated COX-2, and a COX-3 isoform whose RNA is derived through the retention of a highly structured, G + C-rich intron 1 of the COX-1 gene. As generators of oxygen radicals, lipid mediators, and the pharmacological targets of nonsteroidal anti-inflammatory drugs (NSAIDs), COX enzymes potentiate inflammatory neuropathology in Alzheimer's disease (AD) brain. Because COX-2 is elevated in AD and COX-3 is enriched in the mammalian CNS, these studies were undertaken to examine the expression of COX-3 in AD and in [IL-1beta + Abeta42]-triggered human neural (HN) cells in primary culture. The results indicate that while COX-2 remains a major player in propagating inflammmation in AD and in stressed HN cells, COX-3 may play ancillary roles in membrane-based COX signaling or when basal levels of COX-1 or COX-2 expression persist.     

铜蓝蛋白及纤维粘连蛋白对石英粉尘促进胶原转录的协同作用

本文使用人胶原α_1(Ⅰ)、α_2(Ⅰ)和α_1(Ⅲ)链cDNA探针,采用斑点杂交技术观察了铜蓝蛋白(ceruloplasmin,Cp)和纤维粘连蛋白(fibronectin,Fn)对体外培养的人胚肺成纤维细胞胶原mRNA合成的影响。发现Cp及Fn对成纤维细胞胶原mRNA的合成有促进作用。向培养的成纤维细胞中同时加入石英粉尘和Cp时,其刺激胶原mRNA增长的程度高于单独加入石英粉尘或Cp时的刺激作用。提示Cp对石英粉尘的促纤维化有协同作用。本实验同时证实Fn也具有类似于Cp的促纤维化的协同作用。     

Regulation of Collagen Synthesis in Human Dermal Fibroblasts in Contracted Collagen Gels by Ascorbic Acid, Growth Factors, and Inhibitors of Lipid Peroxidation

Ascorbic acid has been shown to stimulate collagen synthesis in monolayer cultures of human dermal fibroblasts. In the present studies, we examined whether the presence of a collagen matrix influences this response of dermal fibroblasts to ascorbic acid. Fibroblasts and collagen were mixed and allowed to gel and contract for 6 days to form a matrix prior to determining the concentration and time dependence for ascorbic acid to affect collagen synthesis by fibroblasts within the matrix. Collagen synthesis was stimulated at levels at or above 10 μM ascorbic acid and was maximal after 2 days of treatment. This concentration and time dependence is similar to that of cells grown in monolayer cultures. The effects of transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF) were also examined in this model. TGF-β increased and FGF inhibited collagen synthesis in the gels, as has been shown for cells in monolayer cultures. The effects of potential inhibitors of lipid peroxidation induced by ascorbic acid were also examined in these matrices and compared to previous results obtained in monolayer cultures. Propyl gallate, cobalt chloride, α,α-dipyridyl, and α-tocopherol inhibited the ascorbic acid-mediated stimulation of collagen synthesis while mannitol had no effect. Natural retinoids inhibited total protein synthesis without the specific effect on collagen synthesis that was seen in monolayer cultures. These results indicate that ascorbic acid stimulates collagen synthesis in fibroblasts grown in a collagen matrix in a manner similar to that found in monolayer cultures. In contracting collagen gels, however, the magnitude of the effect is less and retinoids do not specifically inhibit collagen synthesis.     

遗传性癫痫易感大鼠脑内NMDAR1基因表达

N-甲基-D天门冬氨酸(NMDA)受体与癫痫及癫痫易感性的形成密切相关. 以遗传性癫痫易感大鼠P77PMC为研究对象, 通过RNA印迹杂交检测,NMDA受体一型亚单位(NMDAR1)mRNA在惊厥后不同脑区表达, 结果显示: P77PMC大鼠惊厥后, 大脑皮层、海马、皮层下、下丘NMDAR1 mRNA表达呈时间依赖性增加;比较惊厥即刻与惊厥后24 h, 四个脑区NMDAR1 mRNA分别增加了111%、113%、165%和202%. 提示NMDA受体 亚单位受惊厥活动调控,并参与惊厥的发生、发展及惊厥后突触结构的重建.     

A Selenium-Enriched Ziyang Green Tea Polysaccharide Induces Bax-Dependent Mitochondrial Apoptosis and Inhibits TGF-β1-Stimulated Collagen Expression in Human Keloid Fibroblasts via NG2 Inactivation

Keloids are fibroproliferative disorders characterized by the overabundant deposition of extracellular matrix (ECM), especially collagen and overgrowth of scar tissue in response to cutaneous injury. In this study, we isolated a selenium (Se)-containing polysaccharide (Se-ZGTP-I) from Ziyang green tea and explored its potential therapeutic effects on keloid fibroblasts formation. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and annexin V/propidium iodide (PI) staining assays demonstrated that Se-ZGTP-I or neuron-glia 2 (NG2) short hairpin RNA (shRNA) significantly inhibited proliferation of human keloid fibroblasts via induction of apoptosis. Besides, the activation of caspase-3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP) were observed in keloid fibroblasts following Se-ZGTP-I (200 and 400 μg/ml) or NG2 shRNA treatment. Moreover, Western blotting analysis showed that treatment of keloid fibroblasts with Se-ZGTP-I (200 and 400 μg/ml) or NG2 shRNA resulted in an increase of pro-apoptotic protein Bax expression and a decrease in expression levels of anti-apoptotic protein Bcl-2 and NG2. In addition, type I collagen biosynthesis and protein expression in keloid fibroblasts following TGF-β1 stimulation were decreased by Se-ZGTP-I (200 and 400 μg/ml) or NG2 shRNA management. Current findings imply that Se-ZGTP-I has a therapeutic potential to intervene and prevent keloid formation and other fibrotic diseases.     

CTGF Increases IL-6 Expression in Human Synovial Fibroblasts through Integrin-Dependent Signaling Pathway

Background

Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. CTGF is abundantly expressed in osteoarthritis (OA). However, the relationship between CTGF and IL-6 in OA synovial fibroblasts (OASFs) is mostly unknown.

Methodology/Principal Findings

OASFs showed significant expression of CTGF, and expression was higher than in normal SFs. OASFs stimulation with CTGF induced concentration-dependent increases in IL-6 expression. CTGF mediated IL-6 production was attenuated by αvβ5 integrin neutralized antibody and apoptosis signal-regulating kinase 1 (ASK1) shRNA. Pretreatment with p38 inhibitor (SB203580), JNK inhibitor (SP600125), AP-1 inhibitors (Curcumin and Tanshinone IIA), and NF-κB inhibitors (PDTC and TPCK) also inhibited the potentiating action of CTGF. CTGF-mediated increase of NF-κB and AP-1 luciferase activity was inhibited by SB203580 and SP600125 or ASK1 shRNA or p38 and JNK mutant.

Conclusions/Significance

Our results suggest that CTGF increased IL-6 production in OASFs via the αvβ5 integrin, ASK1, p38/JNK, and AP-1/NF-κB signaling pathways.