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Purpose: 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., Materials and Methods: 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., Results: 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., Conclusion: 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.

The extracellular matrix (ECM) regulates cell behavior through signal transduction and provides a suitable place for cell survival. As one of the major components of the extracellular matrix, type I collagen is involved in regulating cell migration, proliferation and differentiation. We present a system in which 3T3-L1 preadipocyte cells are induced for adipogenic differentiation on type I collagen coated dishes. Our previous study has found that type I collagen inhibits adipogenic differentiation via YAP activation. Here we further reveal that type I collagen inactivates autophagy by up-regulating mTOR activity via the YAP pathway. Under collagen-coating conditions, co-localization of lysosomes with mTOR was increased and the level of downstream protein p-S6K was elevated, accompanied by a decrease in the level of autophagy. Autophagy is negatively correlated with adipogenesis under type I collagen coating. Through the YAP-autophagy axis, type I collagen improves glycolipid metabolism accompanied by increased mitochondrial content, enhanced glucose uptake, reduced release of free fatty acids (FFAs) and decreased intracellular lipid accumulation. Our findings provide insight into the strategy for dealing with obesity: Type I collagen or the drugs with inhibitory effects on autophagy or YAP, have a potential to accelerate the energy metabolism of adipose tissue, so as to better maintain the homeostasis of glucose and lipids in the body, which can be used for achieving weight loss., (Copyright © 2022. Published by Elsevier B.V.)

Type I collagen (collagen I) is the most abundant component of the extracellular matrix (ECM) in the pancreas. We previously reported that collagen I-coated culture dishes enhanced proliferation of rat pancreatic β cell line, INS-1 cells, via up-regulation of β-catenin nuclear translocation. In this study, we further investigated the effects of collagen I on insulin production of INS-1 cells. The results indicate that insulin synthesis as well as cell proliferation is increased in the INS-1 cells cultured on the dishes coated with collagen I. Up-regulation of insulin-like growth factor 1 receptor (IGF-1R) on the INS-1 cells cultured on the collagen-coated dishes is involved in up-regulation of cell proliferation and increase of insulin biosynthesis; however, up-regulation of insulin secretion in the INS-1 cells on collagen I-coated dishes was further enhanced by inhibition of IGF-1R. Autophagy of INS-1 cells on collagen I-coated dishes was repressed via IGF-1R upregulation, and inhibition of autophagy with 3MA further enhanced cell proliferation and insulin biosynthesis but did not affect insulin secretion. E-cadherin/β-catenin adherent junction complexes are stabilized by autophagy. That is, autophagy negatively regulates the nuclear translocation of β-catenin that leads to insulin biosynthesis and cell proliferation. In conclusion, IGF-1R/downregulation of autophagy/nuclear translocation of β-catenin is involved in collagen I-induced INS-1 cell proliferation and insulin synthesis.

Extracellular matrix (ECM) has a marked influence on adipose tissue development. Adipose tissue formation is initiated with proliferation of preadipocytes and migration before undergoing further differentiation into mature adipocytes. Previous studies showed that collagen I (col I) provides a good substratum for 3T3-L1 preadipocytes to grow and migrate. However, it remains unclear whether and how col I regulates adipogenic differentiation of preadipocytes. This study reports that lipid accumulation, representing in vitro adipogenesis of the 3T3-L1 preadipocytes or the mouse primary adipocyte precursor cells derived from subcutaneous adipose tissue in the inguinal region is inhibited by the culture on col I, owing to downregulation of adipogenic factors. Previous study shows that col I enhances 3T3-L1 cell migration via stimulating the nuclear translocation of yes-associated protein (YAP). In this study, we report that downregulation of YAP is associated with in vitro adipogenesis of preadipocytes as well as with in vivo adipose tissue of high-fat diet fed mice. Increased expression of YAP in the cells cultured on col I-coated dishes is correlated with repression of adipogenic differentiation processes. The inactivation of YAP using YAP inhibitor, verteporfin, or YAP small-interfering RNA enhanced adipogenic differentiation and reversed the inhibitory effect of col I. Activation of YAP either by the transfection of YAP plasmid or the silence of large tumor suppressor 1 (LATS1), an inhibitory kinase of YAP, inhibited adipogenic differentiation. The results indicate that col I inhibits adipogenic differentiation via YAP activation in vitro., (© 2019 Wiley Periodicals, Inc.)

Skeletal muscle regeneration is a complicated process, requiring the proliferation, migration and differentiation of myoblasts whose processes are highly regulated by the extracellular matrix (ECM) surrounding the muscle tissues in vivo. However, the effects of respective ECM components on the regulation of myoblast behaviors are unknown. In this study, we report on the effect of collagen I, a major ECM component in muscle tissue and a popular food supplement, on mouse C2C12 myoblast proliferation, migration and differentiation as well as the underlying mechanisms. Collagen I (col 1) enhances the migration and myogenic differentiation of C2C12 cells, but has no effect on cell proliferation. Col I significantly promotes the production and release of interleukin-6 via nuclear translocation of nuclear factor κB (NF-κB) p65. The release of IL-6 plays a critical role in the col I-enhanced migration and differentiation of C2C12 cells. Furthermore, col I increases phosphorylation of focal adhesion kinase (FAK) that is involved in the nuclear translocation of NF-κB p65. Collectively, col I enhances the migration and differentiation of C2C12 cells through IL-6 release induced by FAK/NF-κB p65 activation.

Glycosylation in type I collagen occurs as O-linked galactosyl- (G-) lesser and glucosylgalactosyl-hydroxylysine (GG-Hyl); however, its biological significance is still not well understood. To investigate the function of this modification in bone, we have generated preosteoblast MC3T3-E1 (MC)-derived clones, short hairpin (Sh) clones, in which Glt25d1 gene expression was stably suppressed. In Sh clones, the GLT25D1 protein levels were markedly diminished in comparison to controls (MC and those transfected with the empty vector). In Sh collagen, levels of both G- and GG-Hyl were significantly diminished with a concomitant increase in the level of free-Hyl. In addition, the level of immature divalent cross-links significantly diminished while the level of the mature trivalent cross-link increased. As determined by mass spectrometric analysis, seven glycosylation sites were identified in type I collagen and the most predominant site was at the helical cross-linking site, α1-87. At all of the glycosylation sites, the relative levels of G- and GG-Hyl were markedly diminished, i.e., by ∼50-75%, in Sh collagen, and at five of these sites, the level of Lys hydroxylation was significantly increased. The collagen fibrils in Sh clones were larger, and mineralization was impaired. These results indicate that GLT25D1 catalyzes galactosylation of Hyl throughout the type I collagen molecule and that this modification may regulate maturation of collagen cross-linking, fibrillogenesis, and mineralization.

Background: Extracellular matrix (ECM) comprising the environments of multicellular society has a dynamic network structure. Collagen is one of the ubiquitous components of ECM. Collagen affects the inflammatory response by regulating the release of pro-inflammatory cytokines from cells. Gelatin, denatured collagen found temporally in tissues, is supposed to be pathophysiologically involved in tissue remodeling, inflammation caused by tissue damage. Previous reports indicate that, phorbol myristate (PMA)-stimulated human U937 (lymphoma cell line) cells that are often used as macrophage-like cells, show cell aggregations when cultured on type I collagen (col I) or gelatin-coated dishes, accompanying the changes of production and release of proinflammatory factors. However, it still remains to be examined whether collagen and gelatin affects normal macrophages as well., Aim: This study aims to investigate the effect of col. I, the main component of collagenous protein and its denatured product, gelatin, on mouse peritoneal macrophages (MPMs)., Methods: MTT assay, flow cytometric analysis of ROS, biochemical detection of antioxidant levels, ELISA assay, and western blot were used., Results: MPMs formed multicellular aggregates on col. I - and gelatin-coated dishes with a concentration- and time-dependent manner. Further studies showed that the culture on col. I and gelatin up-regulated the protein expression and secretion of pro-inflammatory molecules such as IL-1β, TNFα and prostaglandin E 2 (PGE 2 ) in MPMs. The levels were higher in the cells on gelatin than those on col. I. ROS levels are significantly increased in the cells cultured on both col. I- and gelatin-coated dishes, accompanying decreased levels of antioxidant enzyme catalase (CAT) and anti-oxidant glutathione (GSH), and enhanced nuclear translocation of NF-κB., Conclusion: Col I - or gelatin-coated culture induced the formation of multicellular aggregates and increased production of NF-κB-associated pro-inflammatory molecules in MPMs through up-regulation of ROS levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structure N ω -(carboxymethyl)arginine (CMA) is produced by oxidation in glycated collagen, accounting for one of the major proteins detected in biological samples. In this study, we investigated the mechanism by which CMA is generated in collagen and detected CMA in collagen-rich tissues. When various protein samples were incubated with glucose, the CMA content, detected using a monoclonal antibody, increased in a time-dependent manner only in glycated collagen, whereas the formation of N ε -(carboxymethyl)lysine (CML), a major antigenic AGE, was detected in all glycated proteins. Dominant CMA formation in glycated collagen was also observed by electrospray ionization-liquid chromatography-tandem mass spectrometry (LC-MS/MS). During incubation of glucose with collagen, CMA formation was enhanced with increasing glucose concentration, whereas it was inhibited in the presence of dicarbonyl-trapping reagents and a metal chelator. CMA formation was also observed upon incubating collagen with glyoxal, and CMA was generated in a time-dependent manner when glyoxal was incubated with type I-IV collagens. To identify hotspots of CMA formation, tryptic digests of glycated collagen were applied to an affinity column conjugated with anti-CMA. Several CMA peptides that are important for recognition by integrins were detected by LC-MS/MS and amino acid sequence analyses. CMA formation on each sequence was confirmed by incubation of the synthesized peptides with glyoxal and ribose. LC-MS detected CMA in the mouse skin at a higher level than other AGEs. Furthermore, CMA accumulation was greater in the human aorta of older individuals. Overall, our study provides evidence that CMA is a representative AGE structure that serves as a useful index to reflect the oxidation and glycation of collagen., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2019 Sho Kinoshita et al.)

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.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy. Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Leather produced from crocodile, alligator, and caiman skin is widely used in the fashion industry. Crocodilian leather is generally more expensive than mammalian leather, and the value greatly differs even between the crocodilian species. However, inappropriate labeling of the animal source on leather products sometimes arises from accidental or fraudulent substitution, which is difficult to unambiguously detect by existing methods. In the present study, animal source identification of crocodilian leather was carried out using type I collagen-derived marker peptides generated after dechroming, heat denaturation, and trypsin digestion. Definitive discrimination between the three crocodilian species and also a related species, lizard, was achieved based on the detection patterns of selected six marker peptides, determined by LC-MS. Furthermore, powdering of the leather samples enabled a reduction in the sample amount required and allowed the elimination of the dechroming step. Approximately 100 μg of powder was taken from commercial leather watch straps by filing, resulting in only slight damage to the undersides of the straps. The animal sources of the crocodilian products and also a crocodile-embossed calf product were successfully identified using a combination of the crocodilian marker peptides and previously established mammalian marker peptides. This semi-nondestructive species identification method is not only useful for certification of leather products but also for monitoring of international trade of leather and skin.

The extracellular matrix (ECM) is a major biomechanical environment for all cells in vivo, and tightly controls wound healing and cancer progression. Type I collagen (Col I) is the most abundant component in ECM and plays an essential role for cell motility control and migration beyond structural support. Our previous results showed that Col I increased the length of primary cilia and the expression of primary cilia-associated proteins in 3T3-L1 cells. The Hippo/YAP pathway serves as a major integrator of cell surface-mediated signals and regulates key processes for the development and maintenance of tissue functions. In this study, we investigated the role of Hippo/YAP signaling in primary cilia growth of cells cultured on Col I-coated plate, as well as the potential link between primary cilia and migration. At 2-day post-confluence, YAP localization in the nucleus was dramatically increased when the cells were cultured on Col I-coated plate, accompanied by cilia growth. YAP inhibitor verteporfin repressed the growth of primary cilia as well as the expressions of ciliogenesis-associated proteins in confluent 3T3-L1 cells cultured on Col I-coated plate. Moreover, knockdown of either YAP or IFT88, one of the ciliogenesis-associated proteins, reversed the migration of confluent 3T3-L1 cells promoted by Col I-coating. In conclusion, activation of YAP pathway by Col I-coating of culture plate for confluent 3T3-L1 cells is positively associated with the primary cilia growth, which eventually results in promoted migration.

Preadipocyte migration is a fundamental and important process for the development of tissue organization, especially in the development of primitive adipose tissue and adipocyte tissue wound healing. However, excessive migration may result in abnormal development and fibrosis-related diseases such as hypertrophic scar. We previously reported that type I collagen (collagen I) enhanced migration of 3T3-L1 preadipocytes via phosphorylation and/or acetylation of NF-κB p65, and the enhanced cell migration is repressed by silibinin treatment through sirt1. It is known that sirt1 has an ability to deacetylate acetylated NF-κB p65, but little is known about the effect of sirt1 on phosphorylated NF-κB p65. This study aims to examine the potential effect of sirt1 on the regulation of phosphorylated NF-κB p65 and the underlying mechanism. Autophagy is involved in many physiological and pathological processes, including regulation of cell migration as well as in cellular homeostasis. The present study demonstrates that silibinin induces autophagy in a dose-dependent manner in 3T3-L1 cells. Autophagy is under the regulation of sirt1/AMPK pathway, and inhibits collagen I-enhanced migration of 3T3-L1 cells through negative regulation of NF-κB p65 phosphorylation but not acetylation. The expression of peroxisome proliferator-activated receptor α (PPARα) is up-regulated with silibinin accompanying up-regulation of autophagy through activating sirt1 in 3T3-L1 cells. Taken together, these findings indicate that silibinin-induced autophagy is mediated by up-regulation of PPARα-sirt1-AMPK, contributing to repression of type I collagen-enhanced migration in murine 3T3-L1 preadipocytes through down-regulation of phosphorylated NF-κB p65.

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.

In organ fibrosis, mechanical stress and transforming growth factor beta-1 (TGF-β1) promote differentiation into myofibroblast from mesenchymal cells, leading to extracellular matrix (ECM) remodeling or active synthesis, deposition or degradation of ECM components. A major component of ECM, type I collagen (col I) triple helical molecules assemble into fibrils or are denatured to gelatin without triple-helicity in remodeling. However, whether changes of ECM components in remodeling have influence on mesenchymal cell differentiation remains elusive. This study adopted three states of collagen I existing in ECM remodeling: molecular collagen, fibrillar collagen and gelatin to see what are characteristics in the effects on two cell lines of mesenchymal origin, murine 3T3-L1 embryonic fibroblast and murine C2C12 myoblasts. The results showed that all three forms of collagen I were capable of inducing these two cells to differentiate into myofibroblasts characterized by increased expression of alpha-smooth muscle actin (α-SMA) mRNA. The expression of α-SMA is positively regulated by TGF-β1. Nuclear translocation of Yes-associated protein (YAP) is involved in this process. Focal adhesion kinase (FAK) is activated in the cells cultured on molecular collagen-coated plates, contributing to YAP activation. On the other hand, in the cells cultured on fibrillar collagen gel or gelatin-coated plates, oxidative stress but not FAK induce YAP activation. In conclusion, the three physicochemically distinct forms of col I induce the differentiation of mesenchymal cells into myofibroblasts through different pathways., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

The present study is aimed to investigate the effect of collagen I on U937 cells, human monocyte-like histiocytic lymphoma cell line. Differentiation of U937 cells was induced by phorbol ester (PMA) treatment. The cells were cultured on the collagen I-coated plate. PMA-stimulated U937 cells formed multicellular aggregates on collagen I-coated surface, whereas PMA-unstimulated cells kept themselves away off each other. Moreover, the levels of reactive oxygen species (ROS) and productions of pro-inflammatory cytokines such as IL-1β, TNFα and PGE2, pro-inflammatory mediator, were down-regulated in differentiated U937 cells cultured on collagen I-coated dishes. However, collagen I did not influence the capacity of E. coli phagocytosis. Cell aggregation as well as the down-regulation of IL-1β, TNFα and PGE2 caused by the culture on collagen I-coated surface were suppressed by ROS donor, tert-butylhydroperoxide (tBHP). The sizes of cell aggregates became bigger in differentiated U937 cells by treatment with ROS scavengers such as N-acetylcysteine (NAC), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). In conclusion, collagen I-coated culture induces the differentiated U937 cells to form cell aggregates and decreases the production of pro-inflammatory cytokines through down-regulating ROS generation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Primary cilia are microtubule-based organelles that extend from nearly all vertebrate cells. Abnormal ciliogenesis and cilia length are suggested to be associated with hypertension and obesity as well as diseases such as Meckel-Gruber syndrome. Extracellular matrix (ECM), comprising cellular microenvironment, influences cell shape and proliferation. However, influence of ECM on cilia biogenesis has not been well studied. In this study we examined the effects of type I collagen (col I), the major component of ECM, on primary cilia growth. When cultured on collagen-coated dishes, confluent 3T3-L1 cells were found to exhibit fibroblast-like morphology, which was different from the cobblestone-like shape on non-coated dishes. The level of autophagy in the cells cultured on col I-coated dishes was attenuated compared with the cells cultured on non-coated dishes. The cilia of the cells cultured on col I-coated dishes became longer, accompanying increased expression of essential proteins for cilia assembly. Transfection of the siRNA targeting microtubule-associated protein light chain 3 (LC3) further enhanced the length of primary cilia, suggesting that col I positively regulated cilia growth through inhibition of autophagy. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy in our previous study on primary cilia, was down-regulated with col I. 3T3-L1 cells treated with the siRNA against HDAC6 reduced the autophagy level and enhanced collagen-induced cilia elongation, implying that HDAC6 was involved in mediating autophagy. In conclusion, col I promotes cilia growth through repressing the HDAC-autophagy pathway that can be involved in the interaction between primary cilia and col I., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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.

Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.

Bovine type I collagen consists of two α1 and one α2 chains, containing the internal triple helical regions and the N- and C-terminal telopeptides. In industries, it is frequently digested with porcine pepsin to produce a triple helical collagen without the telopeptides. However, the digestion mechanism is not precisely understood. Here, we performed a mass spectrometric analysis of the pepsin digest of the N-terminal telopeptide pQLSYGYDEKSTGISVP (1-16) in the α1 chain. When purified collagen was digested, pQLSYGY (1-6) and pQLSYGYDEKSTG (1-12) were identified, while DEKSTG (7-12) was not. When the N-terminal telopeptide mimetic synthetic peptide pQLSK(MOCAc)GYDEKSTGISK(Dnp)P-NH 2 was digested, pQLSK(MOCAc)GYDEKSTG (1-12) and ISK(Dnp)P-NH 2 (13-16) were readily identified, pQLSK(MOCAc)GY (1-6) and DEKSTGISK(Dnp)P-NH 2 (7-16) were weakly detected, and DEKSTG (7-12) was hardly identified. These results suggest that pepsin preferentially cleaves Tyr6-Asp7 and less preferentially Gly12-Ile13. They also suggest that the former cleavage requires native collagen structure, while the latter cleavage does not.

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5-18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro(707) and newly identified sites at Pro(716) and Pro(719), at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N- and C-terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N- and in the C-terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N-terminal and in the C-terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS-PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the Km and kcat values increased with increasing temperature (30 to 65 °C), although the kcat /Km values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the Km and kcat values increased with decreasing pH, and the kcat /Km values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin., (© 2015 Institute of Food Technologists®)

Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We recently developed a novel method for analysis of collagen O-glycosylations, which include galactosyl-hydroxylysine (GHL) and glucosyl-galactosyl-hydroxylysine (GGHL), using hydrazide chemistry (Taga, Y., Mol. Cell. Proteomics 2012, 11 (6), M111.010397). Here we investigated an overglycosylation model of collagen produced by cultured skin fibroblasts from osteogenesis imperfecta (OI) patients using this method. Many GHL/GGHL sites were identified in normal and OI type I collagens by LC-MS analysis after the glycopeptide purification procedure. Further, relative quantification was performed on each identified glycopeptide using stable isotope labeling by amino acids in cell culture (SILAC). Significant increases of GGHL were observed at respective glycosylation sites of type I collagen in OI, whereas an OI-specific glycosylation site was not found. These results demonstrated that the overglycosylation of type I collagen proceeds only at specific sites, resulting in accumulation of GGHL, rather than because of an increase of nonspecific glycosylation. Although the roles of collagen O-glycosylations in OI and even in normal conditions are still incompletely understood, the location of GHL/GGHL in the collagen sequence is suggested to be important for their functions.

In recent years, glycopeptide purification by hydrazide chemistry has become popular in structural studies of glycoconjugates; however, applications of this method have been almost completely restricted to analysis of the N-glycoproteome. Here we report a novel method for analyzing O-glycosylations unique to collagen, which are attached to hydroxylysine and include galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine. We established a hydrazide chemistry-based glycopeptide purification method using (1) galactose oxidase to introduce an aldehyde into glycopeptides and (2) formic acid with heating to elute the bound glycopeptides by cleaving the hydrazone bond. This method allows not only identification of O-glycosylation sites in collagen but also concurrent discrimination of two types of carbohydrate substitutions. In bovine type I and type II collagens, galactosyl-hydroxylysine /glucosyl-galactosyl-hydroxylysine -containing peptides were specifically detected on subsequent comprehensive liquid chromatography (LC)/MS analysis, and many O-glycosylation sites, including unreported ones, were identified. The position of glycosylated hydroxylysine, which is determined by our unambiguous and simple method, could provide insight into the physiological role of the modifications.

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.

In the present study, we evaluated the antioxidant effects of a pepsin-treated novel collagen peptide (P-NCP) on reactive oxygen species (ROS) such as hydroxyl radical (HO(•)), superoxide anion radical (O(2)(•-)), and singlet oxygen ((1)O(2)), and the effects on cell viability after ultraviolet ray (UV) irradiation of human fibroblasts. We confirmed, using electron spin resonance, that P-NCP directly inhibited HO(•) and (1)O(2). Furthermore, addition of P-NCP to fibroblasts inhibited cell death induced by UVA (400-315 nm) irradiation in a dose-dependent manner. In addition, the antioxidant effect on (1)O(2) was observed in the peptide fractions rich in Gly, Pro, Hyp, Glu, Ala, and Arg. We found that Gly, Hyp, Glu, and Ala directly scavenged (1)O(2). These results indicated that a peptide sequence including Gly, Hyp, Glu, and Ala could play a key role in the antioxidant effects of P-NCP on (1)O(2). It was suggested that P-NCP can inhibit photo-aging related to ROS owing to its antioxidant effects.

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.

The fibrillar collagen I gel induced the formation of numerous dendritic cell-like protrusions (cell spikes) from the cell body, whereas monomeric collagen I induced typical cell spreading with filopodia and lamellipodia in skin fibroblasts. Peripheral, not central stress fibers appeared upon adhesion to fibrillar collagen gel, whereas both types of fibers were evident upon adhesion to monomeric collagen. Microtubules and vimentin filaments were elongated inside stress fibers along the terminal tip of cell spikes. Spike formation was totally inhibited by nocodazole and severely delayed by cytochalasin D. This suggests that cell spike formation is dependent on microtubules rather than on F-actin. We then investigated the intracellular signaling responsible for cytoskeleton organization to identify the key factor that induces cell spike morphology. During cell spike formation, FAK and CAS were activated. More CAS was activated in cells on fibrillar collagen gel than on the monomeric form, whereas FAK was activated to the same level on either. At 90 min of culture, Rac1 was activated in cells on monomeric collagen I, whereas Cdc42, Rac1 and RhoA were activated in cells on fibrillar collagen gel. These results suggest that microtubule organization via CAS and small GTPases is important for the cell spike formation that is involved in collagen gel contraction and in wound retraction in skin.

Cultured human foreskin keratinocytes (HFKs) adhere to and grow on nonfibrous collagen via integrin alpha2beta1. During incubation, the receptors used for adhesion are changed to integrins alpha3beta1 and alpha6beta4 and those receptors bind to laminin 5 which is deposited by keratinocytes themselves. In this report, we examined the behaviors of HFKs and transformed keratinocytes on collagen fibril gels. These cells adhered to and spread on collagen gels using integrin alpha2beta1. After several hours on collagen gels, however, cells became round and apoptosis occurred. The behavior of keratinocytes contrasted to that of fibroblasts that grew well even on collagen gel. At the point of apoptosis, integrins alpha2beta1 and alpha3beta1 were not found in the contact region of HFKs. Also, deposition of laminin 5 on collagen gel was not found despite the synthesis of mRNA for laminin 5 and laminin 10/11, while soluble laminin 5 protein is readily detectable. Phosporylation of Akt, which is known as a survival signal, was detected in HFKs cultured on coated collagen; however, the protein level and signals of Akt were dramatically decreased on collagen gel after 1 day of culture. These results indicate that collagen gel has different effects than nonfibrous collagen on HFKs and transformed keratinocytes and the interactions of integrin alpha3beta1 and laminin 5/10/11 are indispensable for maintenance of keratinocyte adhesion and survival.