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Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy. [ABSTRACT FROM AUTHOR]

Type V collagen (collagen V) is one of the important components of extracellular matrix (ECM) in pancreas. We previously reported that pre-coating collagen V on the culture dishes enhanced insulin production in INS-1 rat pancreatic β cells. In this study, we investigate the underlying mechanism. Insulin biosynthesis and secretion are both increased in INS-1 cells cultured on collagen V-coated dishes, accompanied by the reduced nuclear translocation of Yes-associated protein (YAP), a transcriptional co-activator. YAP, the downstream effector of Hippo signaling pathway, plays an important role in the development and function of pancreas. Inhibition of YAP activation by verteporfin further up-regulates insulin biosynthesis and secretion. Silencing large tumor suppressor (LATS), a core component of Hippo pathway which inhibits activity of YAP by phosphorylation, by siRNA transfection inhibits both insulin biosynthesis and secretion. In the present study, the protein level of insulin-like growth factor 1 receptor (IGF-1 R), detected as the upstream molecule of YAP, is reduced in the INS-1 cells cultured on the dishes coated with collagen V. The silencing of IGF-1 R by siRNA transfection further enhances insulin biosynthesis and secretion. IGF-1 treatment reduces collagen V–induced up-regulation of insulin biosynthesis and secretion, accompanying the increased nuclear YAP. Inhibition of IGF-1 R/YAP signal pathway is involved in collagen V–induced insulin biosynthesis and secretion in INS-1 cells. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) plays an important role in tissue repair, cell proliferation, and differentiation. Our previous study showed that collagen I and collagen V differently regulate the proliferation of rat pancreatic β cells (INS-1 cells) through opposite influences on the nuclear translocation of β-catenin. In this study, we investigated the β-catenin pathway in INS-1 cells on dishes coated with collagen I or V. We found that nuclear translocation of the transcription factor Yes-associated protein (YAP) was enhanced by collagen I and suppressed by collagen V, but had no effect on INS-1 cell proliferation. Morphologically, INS-1 cells on collagen V-coated dishes showed stronger cell-to-cell adhesion, while the cells on collagen I-coated dishes showed weaker cell-to-cell adhesion in comparison with the cells on non-coated dishes. E-cadherin played an inhibitory role in the proliferation of INS-1 cells cultured on collagen I or collagen V coated dishes via regulation of the nuclear translocation of β-catenin. Integrin β1 was enhanced with collagen I, while it was repressed with collagen V. The integrin β1 pathway positively regulated the cell proliferation. Inhibition of integrin β1 pathway restored the protein level of E-cadherin and inhibited the nuclear translocation of β-catenin in the cells on collagen I-coated dishes, but no effect was observed in the cells on collagen V-coated dishes. In conclusion, collagen I enhances the proliferation of INS-1 cells via the integrin β1 and E-cadherin/β-catenin signaling pathway. In INS-1 cells on collagen V-coated dishes, both integrin β1 and E-cadherin/β-catenin signal pathways are involved in the inhibition of proliferation. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) molecules have multiple functions; prevention of cytotoxicity, provision of mechanical support, cell adhesive substrates and structural integrity in addition to mediation of cellular signaling. In this study, we report that the proliferation of INS-1 cells cultured on collagen I-coated dishes is enhanced, but it is inhibited on collagen V-coated dishes. Inhibitory proliferation on collagen V-coated is not due to apoptosis induction. Silibinin decreases hepatic glucose production and protects pancreatic β-cells, as a potential medicine for type II diabetes. Silibinin up-regulates the proliferation of cells cultured on both collagen I- and V-coated dishes. Collagen-coating regulates gene expression of collagen in a collagen type-related manner. Silibinin increases mRNA expression of collagen I in the cells on collagen I- and V-coated dishes; however, silibinin decreases collagen V mRNA expression on collagen I- and V-coated dishes. Collagen I-coating significantly enhances nuclear translocation of β-catenin, while collagen V-coating reduces it. Differential effects of silibinin on collagen I mRNA and collagen V mRNA can be accounted for by the finding that silibinin enhances nuclear translocation of β-catenin on both collagen I- and V-coated dishes, since phenomenologically nuclear translocation of β-catenin enhances collagen I mRNA but represses collagen V mRNA. These results demonstrate that nuclear translocation of β-catenin up-regulates proliferation and collagen I gene expression, whereas it down-regulates collagen V gene expression of INS-1 cells. Differential gene expressions of collagen I and V by nuclear β-catenin could be important for understanding fibrosis where collagen I and V may have differential effects. [ABSTRACT FROM AUTHOR]

Purpose: Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultured on collagen I-coated dishes express lowered production of pro-inflammatory mediators in parallel through reduced reactive oxygen species (ROS) levels. By contrast, PMA-treated U937 cells on gelatin, the denatured collagen, show enhanced production of pro-inflammatory mediators, mediated by up-regulating autophagy levels. The present study is aimed to investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface. Material and methods: MTT assay, flow cytometric analysis of ROS and autophagy, biochemical detection of antioxidant levels, enzyme-linked immunosorbent assay, and western blot were used. Results: Gelatin-coating increased ROS levels in PMA-treated U937 cells. Increased ROS levels are involved in the regulation of cell aggregation and the release of pro-inflammatory mediators in gelatin-coated culture. These results lead to the query about the crosstalk between the two positive regulators, the autophagy and ROS. Autophagy induction is attenuated by N-acetyl-L-cysteine treatment, but the treatment with autophagy inhibitor, 3-methyladenine, does not affect ROS levels, suggesting ROS are upstream of autophagy in the regulation axis of differentiated U937 cells on gelatin-coated surface. Further study confirmed that upregulation of autophagy was responsible for ROS-induced cell aggregation and production of pro-inflammatory mediators. Conclusion: The results suggest that gelatin-coating promotes the aggregation of PMA-treated U937 cells and the production of pro-inflammatory mediators by ROS-autophagy signaling pathway. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) participate in various cell responses in association with cell proliferation, migration, differentiation, and death. Extracellular matrix (ECM) serves as cellular microenvironments for many kinds of cells, affecting cell activities. However, whether or not ECM influences cellular ROS levels has not been well studied. In this study, cells are cultured on collagen I molecule-coated (mol. coated) dishes and collagen I fibrous gel-covered (gel) dishes to explore their influence on cell behaviours. We found that the levels of ROS in murine 3T3-L1 preadipocytes increased both in cells on mol. coated and those on the gel. Much higher ROS levels were found in the cells cultured on the gel. Cell proliferation and migration were stimulated to opposite directions between the cells on mol. coated and the cells on gel. ROS in a moderate level were positive regulators in the proliferation and migration of cells on mol. coated; however, ROS in a high level served as negative regulators in the cells on gel. These opposite effects on cell proliferation and migration affected by different ROS levels are in parallel with opposite levels of NF-κB p65 activation. [ABSTRACT FROM AUTHOR]

Purpose:Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel.Material and methods:We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy.Results:L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed.Conclusion:NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death. [ABSTRACT FROM AUTHOR]

Purpose: Gelatin has been considered to exist as intermediate substance of collagen catabolism in tissue remodeling or under inflammatory conditions. We have initiated the study on possible biological functions of gelatin that can exist temporally and locally under the conditions of remodeling and inflammationMaterials and methods: To this purpose, we investigated cell proliferation and survival on gelatin-coated dishes and the response to tumor necrosis factor α (TNFα)–induced cytotoxicity in L929 cells. Autophagy level, ATP level, and ROS generation are examined.Results: L929 cells detached from the gelatin-coated dishes and formed multicellular aggregates. TNFα-induced cytotoxicity in L929 cells was inhibited by gelatin-coating culture. The cells on gelatin-coated dishes showed reduced cellular ATP levels and increased adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK) phosphorylation, leading to increased ROS generation and autophagy.Conclusion: This study showed that gelatin-coated culture protected L929 cells from TNFα-induced cytotoxicity and suggested for a possible pathophysiological function of gelatin in regulating cellular functions. [ABSTRACT FROM PUBLISHER]

Medical-grade collagen peptide is used as an additive agent in pharmaceutical formulations; however, it is unknown as to whether the compound exerts antioxidant effects in vitro. In this study, we evaluated the antioxidant effects of medical-grade collagen peptide on reactive oxygen species such as hydroxyl radical, superoxide anion radical and singlet oxygen using electron spin resonance and spin trapping. We confirmed that medical-grade collagen peptide directly inhibited hydroxyl radical generated by the Fenton reaction or by ultraviolet irradiation of hydrogen peroxide, and singlet oxygen. In addition, an antioxidant effect of medical-grade collagen peptide on singlet oxygen was observed in peptide fractions 12-22. The total amount of antioxidant amino acids (Gly, Hyp, Glu, Ala, Cys, Met and His) constituted more than half of the total amino acids in these fractions. These results suggest that the observed antioxidant properties of medical-grade collagen peptide are due to the compound containing antioxidant amino acids. Medical-grade collagen peptide, which is used in pharmaceuticals, and especially in injectables, could provide useful antioxidant properties to protect the active ingredient from oxidation. [ABSTRACT FROM AUTHOR]

Here, we report the behavior of three kinds of human cancer cell lines (Caco-2, MCF-7, HT-1080) on type I collagen substrates, which are in two-dimensional coated collagen or three-dimensional fibrils form. All tested cells on coated collagen adhered and proliferated. However, in the case of collagen fibrils, the proliferation of cancer cells was suppressed. Furthermore, Akt activation, which is known as a cell-survival signal, was inhibited in cells on collagen fibrils. But the activation of ERK1//2 was not completely inhibited. In Caco-2 cells, delay of cell cycle progression and cell death occurred at the same time. Thus, cell division and cell death occurred at equivalent rates on the collagen fibrils, and cell growth seemed to be stopped. These results imply that the fibril form of collagen plays a potential role in inhibiting the growth of cancer cells. [ABSTRACT FROM AUTHOR]

Inherited deficiency for arylsulfatase (Ars) leads to lysosomal storage of sulfated compounds and to serious diseases such as growth retardation, heart failure, and demyelination in the central nervous system. Ars has been regarded as a lysosomal enzyme because of its hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of its enzymatic activity. We previously demonstrated that a large portion of the mammalian arylsulfatase A (ArsA) protein exists on the cell surface of vascular endothelial cells, suggesting that ArsA plays a role in the components of the extracellular matrix. Here we show that ArsA functions as a substrate on which cells adhere and form protrusions. Coating culture plates with recombinant mouse ArsA (rmArsA) stimulates adhesion of human microvascular endothelial cells to the plate followed by the formation of cell protrusions as well as lamellipodia. rmArsA affects the architecture of the cytoskeleton, with a high density of actin filaments localized to peripheral regions of the cells and the extension of bundles of microtubules into the tips of cellular protrusions. rmArsA also affects the distribution pattern of the cell adhesion-associated proteins, integrin α2β1, and paxillin. rmArsA seems to modulate signaling of basic fibroblast growth factor (bFGF) stimulating cytoskeletal rearrangement. We also show that rmArsA tightly binds to sulfated polysaccharides. We suggest that mammalian ArsA plays a role as a novel component of the extracellular matrix. This viewpoint of Ars could be very useful for clarifying the mechanisms underpinning syndromes caused by the deficiency of the function of Ars genes. [ABSTRACT FROM AUTHOR]

We examined the behavior of human foreskin keratinocytes (HFKs) on reconstituted type IV collagen gel. HFKs survived for several days and the upper layer cells expressed a differentiation marker, involucrin. Apoptosis was induced after involucrin expression while cell proliferation was suppressed. On molecular type IV collagen, integrins shifted from α2β1 to α3β1 during HFK culture. On type IV collagen gel, HFKs initially expressed integrin α2β1, and later expressed integrin α3β1 in the presence of α2β1 did not disappear. Using synthetic peptides, we examined integrin α2-mediated adhesion to type IV collagen gel. Addition of synthetic peptide dose-dependently inhibited cell adhesion both on type IV collagen gel and on molecular type IV collagen. On type IV collagen gel, weaker phosphorylation of focal adhesion kinase, paxillin, and Akt was observed compared with the molecular forms. Based on these observations, we think type IV collagen gel is a novel culture substrate that mimics the physiological environment for HFKs. [ABSTRACT FROM AUTHOR]

Keratinocytes adhere and proliferate well on collagen-coated surfaces, but they undergo apoptosis without differentiation on collagen gels according to our past research. In the current studies, we investigated the necessary conditions for keratinocyte survival on fibrous collagen gels. We found that keratinocytes survived on collagen gels when the medium contains elevated levels (1.8 mM) of calcium. Under this high calcium condition, cells formed multicellular colonies and differentiated. Akt was not activated in cells cultured on collagen gels regardless of the calcium concentration, whereas it was activated in cells cultured on nonfibrous collagen. On the other hand, Erk1/2, key kinases of MAPK pathway, were phosphorylated in cells cultured under high calcium condition but not in cells cultured on collagen gels under low calcium condition. The necessity of Erk1/2 activation for keratinocyte survival on collagen gel was confirmed with experiment using U0126, an inhibitor for Erk1/2. These studies show that activation of Akt depends on collagen assembly, whereas activation of Erk1/2 is induced by increased extracellular calcium concentration. Thus, activation of the Erk1/2 by increasing calcium concentration in the incubation medium may compensate for the loss of Akt activation, allowing keratinocyte survival on collagen gels. [ABSTRACT FROM AUTHOR]

Collagen purified from the mantle muscle of the Japanese common squid, Todarodes pacificus , showed autodegradation during incubation under acidic conditions at 25°C, without the addition of exogenous enzymes. This suggests that the collagenolytic proteases bind to collagen tightly through the steps of collagen preparation. Collagenolytic activity also was detected in a crude extract of mantle muscle, and leupeptin and E-64 were observed to inhibit collagenolytic activity within the collagen fraction and muscle extract. We purified these collagenolytic cysteine proteases by leupeptin column chromatography and cellulose acetate membrane electrophoresis. Optimal enzymatic activity was observed at pH 3.5, and collagenolytic activity was completely suppressed at neutral or alkaline pH. The purified enzymes were 28 kDa and 25 kDa in size, and both had gelatinolytic activity, as detected by gelatin zymography, and cut the specific site of denatured collagen α chain. The purified enzymes degraded squid collagen at 25°C, which is 2.5° lower than the temperature at which squid collagen normally denatures; however, the proteases were ineffective at 20°C. Interestingly, the isolated proteases were capable of digesting both squid and bovine gelatin. In this article, we describe collagenolytic cysteine proteases that bind to the collagen of Todarodes pacificus , thereby digesting it by attacking microunfolding regions generated by incubation 2-3°C below the denaturation temperature. [ABSTRACT FROM AUTHOR]